NZ244853A - Antibodies and fragments to icam-1, pharmaceutical composition - Google Patents

Description

2 4 4 8 5 I:., on ^ i' 1 <- : riioriry to. 3©. r . '• .£*»>• Cornp.o'.o • '•- Cb,s: :>.i . .Sf/ ^ Initials - * FuL'.i'.-al.'.: [\0. Jc: rr PATENTS FORM NO. 5 Divisional Application Out of New Zealand Application No: 230775 I1AR1995 ... '.''.i'.'.:- ftflO.

NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION PHARMACEUTICAL COMPOSITIONS We, DANA FARBER CANCER INSTITUTE, a non-profit research hospital of the State of Massachusetts, U.S.A. of 44 Binney Street, Boston, Massachusetts 02115, United States Of America hereby declare the invention, for which we pray that a patent may be granted to us and the method by which it is to be performed, to be particularly described in and by the following statement: (followed by page la) PT0599272 m 2 4 4 8 [ & - la - This invention relates to a pharmaceutical composition comprising an anti-inflammatory agent selected from ICAM-1 or various derivatives and antibodies, with at least one immunosuppressive agent.

This application is a divisional application out of NZ 230775. The discussion of the invention of NZ 230775 is maintained in this specification, for the sake of completeness. However, it should be appreciated that the invention of this application is directed only to the pharmaceutical composition mentioned above and to an anti-inflammatory agent used in the preparation of such compositions.

NZ 230775 relates to intercellular adhesion molecules such as ICAM-1 which are involved in the process through which populations of lymphocytes recognize and adhere to cellular substrates so that they may migrate to sites of inflammation and interact with cells during inflammatory reactions. NZ 230775 additionally relates to ligand molecules capable of binding to such intercellular adhesion molecules, to a screening assay for these ligands, and to uses for the intercellular adhesion molecule, the ligand molecules, and the screening assay.

Leukocytes must be able to attach to cellular substrates in order to properly defend the host against foreign invaders such as bacteria or viruses. An excellent review of the defense system is provided by (followed by page 2) A > / rs L h v 'o . ■) .

E*sen, H.W.. (I*: Mi crop i o 1 ocv. 3rd Ed., Harper i Row, Philadelphia, 3.~ (195CI. :c. 290-295 anc 331-418). They must be aole to attach to encot.nelial cells so that they can migrate from circulation to sites of ongoing inflammation. Furthermore, they must attach to antigen-:resenting cells so that a normal specific immune resoonse can occur, anc finally, tney must attach to appropriate target cells so that lysis cf virally-infected or tumor calls can occur.

Recently, leukocyte surface molecules involved in mediating such attachments were identified using hybricoma technology. Briefly, monoclonal antibodies directed against human T-eel 1s (Oavicnon, D. = t a\, P-oc. Natl. Acad. Sci. USA 78:4325-4539 ( 1931)) anc mouse spleen cells (Springer, I. et aj_. Eur. J. Immunol. 9:301 -306 (1979)) were icentifiec wnich bound to leukocyte surfaces anc inhibited the attachment related functions described above (Springer, T. et al., Fed. Proc. i~:255C-25c3 ( 1935)). The molecules identified by those antibodies were called Mac-1 and Lymphocyte Function-associated Antigen-1 (LFA-1). Mac-1 is a heterodimer found on macrophages, granulocytes and large granular lymphocytes. LFA-1 is a heterodimer found on most lymphocytes (Springer, T.A., et al. Immunol. Rev. 55:111-135 (1382)). These two molecules, plus a third molecule, pI BO,95 (which has a tissue distribution similar to Mac-1) play a role in cellular adhesion (Keizer, G. et al.. Eur. J. Immunol. 15:1142-1147 (1985)).

The above-described leukocyte molecules were found to be members of a related family of glycoproteins (Sanchez-Madrid. F. et al■, J. Exge". Med . I 53:1785-1303 ( 1983): Keizer, G.O. et a 1. . Eur. J. Immunol. !5:li-2-ii-7 (1985)). This glycoprotein family is composed cf heterocimers having one alpha chain and one beta chain. Although tne alpha chain of each of the antigens differed from one another, the beta chain was found to be highly conserved (Sanchez-Madrid, F. et al.. Exoe1". M?d. 153:1785- 1803 ( 1983)). The beta chain of the glycoprotein family (sometimes referred to as "CD1S") was found to have a molecular weight of 95 kc whereas the alpha chains were foun.c to'vary from 150 kc to ISC <d (Sowinger, T. , Fed. Proc. ^£:2550-2553 ',1935;). Althougr. tne 2 4^5 alpha subunits of the membrane proteins do not share the extensive homology shared by the beta subunits, close analysis of 'he alpha subunits of the glycoproteins has revealed that there are substantial similarities between them. Reviews of the similarities between the alpha and beta subunits of the LFA-1 related glycoproteins are provided by Sanchez-Madrid, F. et al.. (J. Exper. Med. 153:586-502 (1583); Exser. Med. 153:1785-1803 (1983)).

A group of individuals has been identified who are unable to express normal amounts of any member of this adhesion protein family on their leukocyte cell surface (Anderson, D.C., et al ., Fed. Proc. ^:2571 -2577 (1SS5); Anderson, D.C., et al ., J. Infect. Pis. 152:663-639 (1935)). Lymphocytes from these patients displayed in vitro defects similar to normal counterparts whose LFA-1 family of molecules had been antagonized by antibodies. Furthermore, these individuals were unable to mount a normal immune response due to an inability of their cells to adhere to cellular substrates (Anderson, O.C., et al.., Fed. Proc. 44:2671-2677 (1985); Anderson, D.C., et al.. J. Infect. Pis. 152:663-689 (1985)). These data show that immune reactions are mitigated vrnen lymphocytes are unable to adhere in a normal fashion due to the lack of functional adhesion molecules of the LFA-1 family.

Thus, in summary, the ability of lymphocytes to maintain the health and viability of an animal requires that they be capable of adhering to other cells (such as endothelial cells). This adherence has been found to require cell-cell contacts which involve specific receptor molecules present on the cell surface of the lymphocytes. These receptors enable a lymphocyte to adhere to other lymphocytes or to endothelial, and other non-vascular cells. The cell surface receptor molecules have been found to be highly related to one another. Humans whose lymphocytes lack these cell surface receptor molecules exhibit chronic and recurring infections, as well as other clinical symptoms including defective antibody responses.

Since lymphocyte adhesion is involved in the process through which foreign tissue is identified and rejected, an understanding of this A 9 3 - 01. W P 2 4 ' ° 5 process is of significant value in the fields of organ transplantation, tissue grafting, allergy and oncology.

SUMMARY OF THIS INVENTION AND THAT OF NZ 230775 NZ 230775 relates to Intercellular Adhesion Molecule-1 (ICAM-1) as well as to its functional derivatives. The invention additionally pertains to antibodies and fragments of antibodies capable of inhibiting the function of ICAM-1, and to other inhibitors of ICAM-i function; and to assays capable of identifying such inhibitors. The invention additionally includes diagnostic and therapeutic uses for all of the above-described molecules.

In detail, the invention includes the intercellular adhesion molecule ICAM-1 or its functional derivatives, which are substantially free of natural contaminants. The invention further pertains to such molecules which are additionally capable of binding to a molecule present on the surface of a lymphocyte.

The invention further pertains to the intercellular adhesion molecule ICAM-1, and its derivatives which are detectably labeled.

The invention additionally includes a recombinant ONA molecule capable of expressing ICAM-1 or a functional derivative thereof.

The invention also includes a method for recovering ICAM-1 in substantially pure form which comprises the steps: (a) solubilizing ICAM-1 from the membranes of cells expressing ICAM-1, to form a solubilized ICAM-1 preparation, (b) introducing the solubilized ICAM-1 preparation to an affinity matrix, the matrix containing antibody caoable of binding to ICAM-1, (c) permitting the ICAM-1 to bind to the antibody of the affinity matrix, (d) removing from the matrix any compound incapable of binding to the antibody and (e) recovering the ICAM-1 in substantially pure form by elutinc the ICAM-1 from the matrix.

A93-01.WP C 3 1729 2 4 8 5 The invention additionally includes an antibody capable of binc.ng to a molecule selected from the group consisting of ICAM-1 anc a functional derivative of ICAM-1. The invention also includes a hybridoma cell capable of producing such an antibody.

The invention further includes a hybridoma cell capable of producing the monoclonal antibody R6-5-D6.

The invention further includes a method for producing a desired hybridoma cell that produces an antibody which is capable of binding to ICAM-1, which comprises: (a) immunizing an animal with a cell expressing ICAM-1, (b) fusing the spleen cells of the animal with a myeloma cell line, (c) permitting the fused spleen and myeloma cells to form antibody secreting hybridoma cells, and (d) screening the hybridoma cells for the desired hybridoma cell that is capable of producing an antibody capable of binding to ICAM-1.

The invention includes as well the hybridoma cell, and the antibody produced by the hybridoma cell, obtained by the above method.

The invention is aUo directed to a method of identifying a non-immunoglobulin antagonist of intercellular adhesion which comprises: (a) incubating a non-immunoglobulin agent capable of being an antagonist of intercellular adhesion with a lymphocyte preparation, the lymphocyte preparation containing a plurality of cells capable of aggregating; (b) examining the lymphocyte preparation to determine whether the presence of the agent inhibits the aggregation of the cells of the lymphocyte preparation; wherein inhibition of the aggregation identifies the agent as an antagonist of intercel1ular adhesion.

The invention is also directed toward a method for treating inflammation resulting from a response of the specific defense system in a mammalian subject which comprises providing to a subject in need of such treatment an amount of an anti-inflammatory agent sufficient to suppress the inflammation; wherein the anti-inflammatory agent is selected from the group consisting of: an antibody capable of bincing A93-01.WP CI 1739 2 4 ■' q 5 to ICAM-1; a fragment of an antibody, the fragment being capable of binding to ICAM-1; ICAM-1; a functional derivative of ICAM-1; and a non-immunoglobulin antagonist of ICAM-1.

The invention further includes the above-described method of treating inflammation wherein the non-immunoglobulin antagonist of ICAM-1 is a non-immunoglobulin antagonist of ICAM-1 other than LFA-1.

The invention is also directed tc a method of suppressing the metastasis of a hematopoietic tumor cell, the cell requiring a functional member of the LFA-1 family for migration, which methoc comprises providing to a patient in need cf such treatment an amount of an anti-inflammatory agent sufficient tc suppress the metastasis; wherein the anti-inflammatory agent is selected from the group consisting of: an antibody capable of bincing to ICAM-1; a fragment of an antibody, the fragment being capable of binding to ICAM-1; ICAM-1; ICAM-1; a functional derivative of ICAM-1; and a non-immunoglobul in antagonist of ICAM-1.

The invention further includes the above-described method of suppressing the metastasis of a ijeaatoooietic Xuaor cell, wherein the non-immunoglobulin antagonist of ICAM-1 is a non-immunoglobul in antagonist of ICAM-1 other than LFA-1.

The invention also includes a method of suppressing the growth of an ICAM-l-exprsssing tumor cell which comorises providing to a patient in need of such treatment an amount of a toxin sufficient to suppress the growth, the toxin being selected from the group consisting of a toxin-derivatized antibody capable of binding to ICAM-1; a toxin-derivatizec fragment of an antibody, the fragment being capable of binding to ICAM-1; a toxin-derivatized member of the LFA-1 family of molecules; and a toxin-derivatized functional derivative of a member of the LFA-1 family of molecules.

The invention is also directed to a method of suppressing the growth of an LFA-l-expressing tumor cell which comprises providing to a patient in need of such treataent• an -amount cf toxin sufficient to suppress such growth, the toxin being selected from the grouo 031789 2 4 4 C 5 3 consisting of a toxin-derivatized ICAM-1; and a toxin-derivatizec functional derivative of ICAM-1.

The invention is further directed toward a method of ciagnosing the presence and location of an inflammation resulting from a response of the specific defense system in a mammalian subject suspected of havinc the inflammation which comprises: (a) administering to the subject a composition containing 3 detectably labeled binding ligand capable of identifying a cell which expresses ICAM-1, and (b) detecting the binding ligand.

The invention additionally provides a method of diagnosing the presence and location of an inflammation resulting from a response of the specific defense system in a mammalian subject suspected of having the inflammation which comprises: (a) incubating a sample of tissue of the subject with a composition containing a detectably labeled binding ligand capable of identifying a cell which expresses ICAM-1, and (b) detecting the binding ligand.

The invention also pertains to a method of diagnosing the presence and location of an ICAM-l-expressing tumor call in a mammalian subject suspected of having such a cell, which comprises: (a) administering to the subject a composition containing a detectaoly labeled binding ligand capable of binding to ICAM-1, the ligand being selected from the group consisting of an antibody and a fragment of an antibody, the fragment being capable of bincing to E CAM -1, and (b) detecting the binding ligand.

The invention also pertains to a method of diagnosing the presence and location of an ICAM-l-expressing tumor cell in a mammalian subject suspectec cf having such a cell, which comprises: (a) incubating a sample of tissue of the subject with a composition containing a detectably labeled binding ligand capable of binding ICAM-1, the ! -icand being selected from group consisting of antibody anc a 0 2 4 4 3 5 fragment of an antibody, the fragment being capable of binding to ICAM-1, and (b) detecting the binding ligand.

The invention also pertains to a method of diagnosing the presence and location of a tumor cell which expresses a member of the LFA-1 family of molecules in a subject suspected of having such a cell, which earneri ses: (a) administering to the subject a composition containing a detectably labeled binding ligand capable of binding to a member of the LFA-1 family of molecules, the ligand being selected from the group consisting of ICAM-1 and a functional derivative of ICAM-1, and (b) detecting the binding ligand.

The invention also pertains to a method of diagnosing the presence and location of a tumor cell which expresses a member of the LFA-1 family of molecules in a subject suspected of having such a cell, which comprises: (a) incubating a sample of tissue of the subject in the presence of a detectably labeled binding ligand capable of binding to a member of the LFA-1 family of molecules, the ligand being selected from the group consisting of ICAM-1 and a functional derivative of ICAM-1, and (b) detecting the binding ligand which is bound to a member of the LFA-1 family of molecules present in the sample of tissue.

On the other hand, this invention relates to a pharmaceutical composition comprising: (a) an anti-inflammatory agent selected from the group consisting of: an antibody caoable of binding to ICAM-1; a fragment of an antibody, the fragment being capable of binding to ICAM-1; ICAM-1; a functional derivative of ICAM-1; and a non-immunoglobulin antagonist of ICAM-1, and (b) at least one immunosuppressive agent selected from the group consisting of: dexamethesone, azathioprine and cyclosporin A.

AS2-01.WP 031739 2 4 4-5 BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows in diagrammatic form the adhesion between a normal anc an LFA-1 deficient cell.

Figure Z shows in diagrammatic form the process of normal/normal ceil achesicn.

Figure 2 shows the kinetics of cellular aggregation in the absence (X) or presence of 50 ng/ml of PMA (0).

Figure - shows coaggregation between LFA-1" and LFA-lr cells. Carboxyf1ucrescein diacetate labeled E3V-transformed cells (104) as cesignated in the figure were mixed with 103 unlabeled autologous cells (solid bars' or JY cells (open bars) in the presence of PMA. After 1.5 h the 1abe*ec cells, in aggregates or free, were enumerated using the cualitative assay of Example 2. The percentage of labeled cells in aggregates is shown. One representative experiment of two is shown.

Figure 5 shows the immunoprecipitation of ICAM-1 and LFA-1 from JY cells. Triton X-100 lysates of JY cells (lanes 1 and 2) or control lysis buffer (lanes 3 and 4) were immunoprecipitatad with antibody capable of binding to ICAM-1 (lanes 1 and 3) or antibodies capable of binding to LFA-1 (lanes 2 and 4). Panel A shows results under reducing conditions; Panel B shows results obtained under non-reducing conditions. Molecular weight standards were run in lane S.

Figure c shows the kinetics of IL-1 and gamma interferon effects on ICAM-1 ex:ression on human dermal fibroblasts. Human dermal fibroblasts were grown to a density of 8 x 10* ceils/0.32 cm^ well. IL-1 (10 U.'Til, closed circles) or recombinant gamma interferon (10 U/'ml, ooen scuares) was added, and at the indicated time, the assay was cooled to ~'C and an indirect binding assay was performed. The standard deviation did not exceed 10%.

Figure 7 shows the concentration dependence of IL-1 and camma interferon effects on ICAM-1. Human dermal fibroblasts were grown to a density of 8 x 10"* cells/0.32 cm^/well. IL - 2 (open circle), recombinant human IL-1 (open square), recombinant mouse IL-1 (solid -93-0!.WP 02I739 2 4 4 8 5 square), recombinant human gamma interferon (solid circles), and recombinant beta interferon (open triangle) were added at the indicated cilution and were incubated for 4 hours (IL-1) or 16 hours (beta and gamma interferon). The indicated results are the means from cuadruplicate determinations; standard deviation did not exceed 10%. figure 8 shows the nucleotide and amino acid sequence of ICAM-1 cDNA. The first ATG is at position 53. Translated sequences corresponding to ICAM-1 tryptic peptides are underlined. The hydrophobic putative signal peptide and transmembrane sequences have a :old underline. N-linked glycosylat i on sites are boxea. The :olyadenylation signal AATAAA at position 2S76 is over-lined. The sequence shown is for the HL-60 cONA clone. The endothelial cell cDNA was sequenced over most of its length and showed only minor ci fferences.

Figure 9 shows the ICAM-1 homologous domains and relationship to the immunoglobulin supergene family. (A) Alignment of 5 homologous domains (D1 -5). Two or more identical residues which aligned are boxed. Residues conserved 2 or more .times in NCAM domains, as well as resides conserved in domains of the sets C2 and CI were aligned with the ICAM-1 internal repeats. The location of the predicted 0 strands in the ICAM-1 domain is marked with bars and lower case letters above the alignments, and the known location of ^-strands in immunoglobulin C comains is marked with bars and capital letters below the alignment. The position of the putative disulfide bridge within ICAM-1 domains is indicated by S-S. (5-D) Alignment of protein domains homologous to ICAM-1 domains; proteins were initially aligned by searching NBRF databases using the FASTP program. The protein sequences are MAG, NCAM, T cell receptor a subunit V domain, IgMjx chain and a-i-B-glycoprotei n.

Figure 10 shows a diagrammatic comparison of the secondary structures of ICAM-1 and MAG.

Figure 11 shows LFA-l-positive ESV-trans farmed 8-lymphoblastoid cells binding to ICAM-1 in planar membranes. -93-01.WP 021789 2 4 4 8 53 - II - Figure 12 shows LFA-l-positive T lympnoblasts and T lymphoma ceils bind to ICAM-1 in plastic-bound vesicles.

Figure 13 shows the inhibition of binding of JY 3-lymphoblastoic cell binding to ICAM-1 in plastic-bound vesicles by pretreatment cf cells or vesicles with monoclonal antibodies.

Figure 14 shows the effect of temperature on binding of T-lymphoblasts to ICAM-1 in plastic-bound vesicles.

Figure 15 shows divalent cation requirements for binding of 7-lympnoblasts to ICAM-1 in plastic-bound vesicles.

Figure 15 shows the effect of anti-adhesion antibodies on tne ability of peripheral blood mononuclear cells to proliferate in response to the recognition of the T-cell associated antigen 0K73. "0KT3" indicates the addition of antigen.

Figure 17 shows the effect of anti-adhesion antibodies on the ability of peripheral blood mononuclear cells to proliferate in response to the recognition of the non-specific T-cell mitogen, concanavalin A. "CONA" indicates the addition of concanavalin A.

Figure 18 shows the effect of anti-adhesion antibodies on the ability of peripheral blood mononuclear cells to proliferate in response to the recognition of the keyhole limpet hemocyanin anticen. The addition of keyhole limpet hemocyanin to the cells is indicated by "KLH." Figure 19 shows the effect of artti-adhesion antibodies on the ability of peripheral blood mononuclear cells to' proliferate in response to the recognition of the tetanus toxoid antigen. The addition of tetanus toxoid antigen to the calls is indicated by "AGN." Figure 20 shows the binding of monoclonal antibodies RR1/1, R6.5, LB2, and CL203 to ICAM-1 deletion mutants.

Figure 21 shows the binding of ICAM-1 deletion mutants to LFA-1.

Figure 22 shows the epitopes recognized by anti-ICAM-1 monoclonal antibodies RR1/1, R6.5, LB2, and CL203.

Figure 23 shows binding capacity of ICAM-1 domain 2 mutants to LFA- 1.

A93-01.WP 021759 Figure 24 shows binding capacity of ICAM-1 domain 3 mutants to LFA- 1.

Figure 25 shows binding capacity of ICAM-1 domain 1 mutants to LFA- 1.

Figure 26 shows the alignment of ICAM amino-terminal domains.

DESCRIPTION OF THE PREFERRED EMBODIMENTS One aspect of NZ 230775 relates to the discovery of a natural binding ligand to LFA-1. Molecules such as those of LFA-1 family, which are involved in the process of cellular adhesion are referred to as "adhesion molecules." The natural binding ligand is designated "Intercellular Aadhesion Molecule-1" or "ICAM-1." ICAM-1 is a 76-97 Kd glycoprotein. ICAM-1 is not a heterodimer. NZ 230775 is directed toward ICAM-1 and its "functional derivatives." In the present application, the term "functional derivative of ICAM-1" excludes the soluble derivatives of ICAM-1 claimed in claim 1 of NZ 230775. A "functional derivative" of ICAM-1 is a compound which possesses a biological activity (either functional or structural) that is substantially similar to a biological activity of ICAM-1. The term "functional derivatives" is intended to include the "fragments," "variants," "analogs," or "chemical derivatives" of a molecule. A "fragment" of a molecule such as ICAM-1, is meant to refer to any polypeptide subset of the molecule. A "variant" of a molecule such as ICAM-1 is meant to refer to a molecule substantially similar in structure and function to either the entire molecule, or to a fragment thereof. A molecule is said to be "substantially similar" to another molecule if both molecules have substantially similar structures or if both molecules possess a similar biological activity. Thus, provided that two molecules possess a similar activity, they are considered variants as that term is used herein even if the structure of one of the molecules not found in the other, or if the sequence of amino acid residues is not identical. An 2 4 4 8 5 3 "analog" of a molecule such as ICAM-1 is meant to refer to a molecule substantially similar in function to either the entire molecule or to a fragment thereof. As used herein, a molecule is said to be a "chemical derivative" of another molecule when it contains additional chemical moieties not normally a part of the molecule. Such moieties may improve the molecule's solubility, absorption, biological half life, etc. The moieties may alternatively decrease the toxicity of the molecule, eliminate or attenuate any undesirable side effect of the molecule, etc. Moieties capable of mediating such effects are disclosed in Remington's Pharmaceutical Sciences (1S80). "Toxin-derivatized" molecules constitute a special class of "chemical derivatives." A "toxin-derivatized" molecule is a molecule (such as ICAM-1 or an antibody) which contains a toxin moiety. The binding of such a molecule to a cell brings the toxin moiety into close proximity with the cell anc thereby promotes cell death. Any suitable toxin moiety may be employed; however, it is preferable to employ toxins such as, for example, the ricin toxin, the diphtheria toxin, radioisotopic toxins, membrane-channel-foraing toxins, etc. Procedures for coupling such moieties to a molecule are well known in the art.

An antigenic molecule such as ICAM-1, or members of the LFA-1 family of molecules are naturally expressed on the surfaces of lymphocytes. Thus, the introduction of such cells into an appropriate animal, as by intraperitoneal injection, etc., will result in the production of antibodies capable of binding to ICAM-1 or members of'the LFA-1 family of molecules. If desired, the serum of such an animal may be removed and used as a source of polyclonal antibodies caoable of binding these molecules. It is, however, preferable to remove splenocytes from such animals, to fuse such spleen cells with a myeloma cell line and to permit such fusion cells to form a hybridoma cell which secretes monoclonal antibodies capable of binding ICAM-1 or members of the LFA-1 family of molecules.

The hybridoma cells, obtained in the manner described above may be screened by a variety of methods to identify desired hybridoma cells A93-01.WP 031733 2 4 4 8 5 3 that secrete antibody capable of binding either to ICAM-1 or to members of the LFA-1 family of molecules. In a preferred screening assay, such molecules are identified by their ability to inhibit the aggregation of Epstein-Barr virus-transformed cells. Antibodies caoable of inhibiting such aggregation are then further screened to determine whether they inhibit such aggregation by binding to ICAM-1, or to a member of the LFA-1 family of molecules. Any means capable of distinguishing ICAM-1 from the LFA-1 family of molecules may be employed in such a screen. Thus, for example, the antigen bound by the antibody may be analyzes as by immunoprecipitation and polyacrylamide gel electrophoresis. If the bound antigen is a member of the LFA-1 family of molecules then tne immunoprecipitated antigen will be found to be a dimer, whereas if the bound antigen is ICAM-1 a single molecular weight species will have been immunoprecipitated. Alternatively, it is possible to distinguish between those antibodies which bind to members of the LFA-1 family of molecules from those which bind ICAM-1 by screening for the ability of the antibody to bind to cells such as granulocytes, which express LFA-1, but not ICAM-1. The ability of an antibody (known to inhibit cellular aggregation) to bind to granulocytes indicates that the antibody is capable of binding tfA-1. The absenc® of such binding is indicative of an antibody capable of recognizing ICAM-1. The ability of an antibody to bind to a cell such as a granulocyte may be detected by means commonly employed by those of ordinary skill. Such means include immunoassays, cellular agglutination, filter binding studies, antibody precipitation, etc.

The anti-aggregation antibodies of NZ 230775 may alternatively be identified by measuring their ability to differentially bind to cells which express ICAM-1 (such as activated endothelial cells), and their inability to bind to cells which fail to express ICAM-1. As will be readily appreciated by those of ordinary skill, the above assays may be modified, or performed in a different sequential order to provide a variety of potential screening assays, each of which is capable of identifying and discriminating between A93-01 .WP o-1 '00 U w » i w «< 2 4 4 8 5 3 antibodies capable of binding to ICAM-1 versus members of the LFA-1 family of molecules.

The anti-inflammatory agents of the present invention may be obtained by natural processes (such as, for example, by inducing an animal, plant, fungi, bacteria, etc., to produce a non-immunoglobu1in antagonist of ICAM-1, or by inducing an animal to produce polyclonal antibodies capable of binding to ICAM-1); by synthetic methods (such as, for example, by using the Merrifield method for synthesizing polypeptides to synthesize ICAM-1, functional derivatives of ICAM-1, or protein antagonists of ICAM-1 (either immunoglobulin or ncn-immunoglobulin)); by hybridoma technology (such as, for example, to produce monoclonal antibodies capable of binding to ICAM-1); or by recombinant technology (such as, for example, to produce the antiinflammatory agents of the present invention in diverse hosts (i.e., yeast, bacteria, fungi, cultured mammalian cells, etc.), or from recombinant plasmids or viral vectors). The choice of which method to employ will depend upon factors such as convenience, desired yield, etc. It is aot necessary to eaqloy o^ily one of the above-described methods, processes, or technologies to produce a particular antiinflammatory agent; the above-described processes, methods, and technologies may be combined in order to obtain a particular antiinflammatory agent.

A. Identification of the LFA-l Binding Partner (ICAM-H 1. Assays of LFA-1-Dependent Aggregation Many Epstein-Barr virus-transformed cells exhibit aggregation. This aggregation can be enhanced in the presence of phorbol esters. Such homotypic aggregation (i.e., aggregation involving only one cell type) was found to be blocked by anti-LFA-1 antibodies (Rothlein. R. et al., J. Exoer. Med. 163:1132-11^3 (1586)), which reference is incorporated herein by reference). Thus, the extant of LFA-1-dependent binding may A93-01,WP r-'"oc 2 4 4 8 5 be determined by assessing the extent of spontaneous or pnorbol ester-dependent aggregate formation.

An agent which interferes with LFA-1-dependent aggregation can be identified through the use of an assay capable of determining whether the agent interferes with either the spontaneous, or the pnorbol ester-dependent aggregation of Epstein-Barr virus-transformed cells. Most Epstein-Barr virus-transformed cells may be employed in such an assay as long as the cells are capable of expressing the LFA-1 receptor molecule. Such cells may be prepared according to the technique of Springer, T.A. et aj_., J. Exoer. Med. 160:1901-1913 ( 198-), which reference is herein incorporated by reference. Although any such cell may be employed in the LFA-1 dependent binding assay of the present invention, it is preferable to employ cells of the JY cell line (Terhost, C.T. et al.. P^cc. Natl. Acad. Sci. USA 73:910 ( 1975)). The calls may be cultivated in any suitable culture medium; however, it is most preferable to culture the cells in RMP! 16*0 culture medium supplemented with 10% fetal calf serum ana 50 Mg/ml gentamycin (Gibco Laboratories, NY). The cells should be cultured under conditions suitable for mammalian cell proliferation (i.e., at a temperature of generally 37*C, in an atmosphere of 5% CO2, at a relative humidity of 95%, etc.). 2. LFA-1 Binds to ICAM-1 Human individuals have been identified whose lymphocytes lack the family of LFA-1 receptor molecules (Anderson, D.C. et a'.. Fed. P-oc. 2671-2677 (1985); Anderson, D.C. et al.. J. Infect. Pis. 152:662-589 (1985)). Such individuals are said to suffer from Leukocyte Adhesion Deficiency (LAD). EBV-transformed cells of such individuals fail to aggregate either spontaneously or in the presence of ohorbol esters in the above-described aggregation assay. When such cells are mixes with LFA-1-expressing cells aggregation was observed (Rothlein, R. et a 1 ■, J. Exoer. Med. 153:1 132-1149 ( 1986)) (Figure 1). Imcortantiy, tr.ese A92-01.WP 02'.739 2 4^85 aggregates failed to form if these cells were incubated in the presence of anti-LFA-1 antibodies. Thus, although the aggregation required LFA-1, the ability of LFA-l-deficient cells tc form aggregates with LFA-1 -containing cells indicated that the LFA-1 binding partner was not LFA-1 but was ratner a previously undiscovered cellular adhesion molecule, -icure 1 shows the mechanism of cellular achesion. 3. Interce 11'j 1 ar Adhesion Molecule-I (IC-M-l) The novel intercel 1 ul ar adhesion molecule ICAM-1 was first identified and partially characterized according to the procedure of Roth1e i n, R. et al. (J. Immunol. 137:1270-1274 (1SS6)), which reference is herein incorporated by reference. To detect the ICAM-1 molecule, monoclonal antibodies were prepared from spleen cells of mice immunized with cells from individuals genetically deficient in LFA-1 expression. Resultant antibodies were screened for their ability to inhibit the aggregation of LFA-l-expressing cells (Figure 2). In detail, the ICAM-i molecule, mice were ixsunizsd with.£SV-transforaed B cells from LAD patients which do not express the LFA-1 antigen. The spleen cells from these animals were subsequently removed, rused with myeloma cells, ana allowed to become monoclonal antibody producing hybridoma cells. EBV-transformed 8 cells from normal individuals which express LFA-1 were then incubated in the presence of the monoclonal antibody of the hybridoma cell in order to identify any monoclonal antibody which was capable of inhibiting the pnorbol ester mediated, LFA-1 dependent, spontaneous aggregation of the EBV-transformed 8 cells. Since the hybridoma cells were derived from cells which had never encountered the LFA-1 antigen no monoclonal antibody to LFA-1 was produced. Hence, any antibody found to inhibit aggregation must he capable of binding to an antigen that, although not LFA-1, participated in the LFA-1 adhesion process. Although any method of obtaining sucn monoclonal antibodies may be enclcyed, it is preferable.to .obtain ICAM-J-binding monoclonal antibodies by immunizing 3AL2/C mice using the routes and schedules A93-01.V? 031739 2 4 4 8 5 described by Rothlein, R. et al. (J. Immunol. 132:1270-1274 (1986)) with Epstein-Barr virus-transformed peripheral blood mononuclear c. lis from an LFA-l-deficient individuals. Such cells are disclosed by Soringer, T.A., et al.. (J. Exoer. Med. 160:1901-1913 (1934)).

In a preferred method for the generation and detection of antibodies capable of binding to ICAM-1, mice are immunized with either E5V-transformed B cells which express both ICAM-1 and LFA-1 or more preferably with INF - act ivated endothelial cells which express ICAM-1 but not LFA-1. In a most 'preferred method for generating hybridoma cells which produce anti-ICAM-1 antibodies, a 3alb/C mouse was sequentially immunized with JY cells and with differentiated U937 cells (ATCC CRL-1593). The spleen cells from such animals are removed, fused with myeloma cells and permitted to .develop into antibody-producing nybriccma cells. The antibodies ane screened for their ability to inhibit the LFA-1 dependent, pnorbol ester induced aggregation of an ESV transformed cell line, such as JY cells, that expresses both the LFA-1 receptor and ICAM-1. As shown by Rothlein, R., et al.. (JL Immunol. 117:1270-1274 (1SS7)), antibodies capable of inhibiting such aggregation are then tested for their ability to inhibit the pnorbol ester induced aggregation of a cell line, such as SKVf3 (Oustin, tt., e* ah, J. Exper. Med. 165:672-592 (1987)) whose ability to spontaneously aggregate in the presence of a phorbol ester is inhibited by antibody capable of binding LFA-1 but is not inhibited by anti-ICAM-1 antibodies. Antibodies capable of inhibiting the phorbol ester induced aggregation of cells such as JY cells, but incapable of inhibiting the pnorbol ester induced aggregation of cells such as SKW3 cells are probably anti-ICAM-1 antibodies. Alternatively, antibodies that are caoable of binding to ICAM-1 may be identified by screening for antibodies which are capable of inhibiting the LFA-1 dependent aggregation of LFA-expression cells (such as JY cells) but are incapable of binding to cells that express LFA-1 but little or no ICAM-1 (suctr as normal granulocytes) or are capable of binding to cells that express ICAM-1 but not LFA-1 (such as TNF-activated endothelial calls). Another A33-C1.WP 031739 i H -i 0 alternative is to immunoprecipitate from cells expressing ICAM-1, LFA-1, or both, using antibodies that innijit the LFA-1 dependent aggregation of cells, such as JY cells, and through SDS-PAGE or an equivalent method determine some molecular characteristic of the molecule precipitated with the antibody. If the characteristic is the same as that of ICAM-1 then the antibody can be assumed to be an anti-I CAM-1 antibody.

Using monoclonal antibodies prepared in the manner described above, the ICAM-1 cell surface molecule was purified, and characterized. ICAM-I was purified from human cells or tissue using monoclonal antibody affinity chromatography. In such a method, a monoclonal antibody reactive with ICAM-1 is coupled to an inert column matrix. Any method of accomplishing such coupling may be employed; however, it is preferable to use the method of Oettgen, H.C. et al.. J. Biol. Chem. 219:1203* (1984)). When a cellular lysate is passed through the matrix the ICAM-1 molecules present are adsorbed and retained by the matrix. By altering the pH or the ion concentration of the column, the bound ICAM-1 molecules may be eluted fron the .column. Although any suitable matrix can be employed, it is preferable to employ sepharose (Pharmacia) as the matrix material. Tne formation of column matrices, and their use in protein purification are well known in the art.

In a manner understood by those of ordinary skill, the above-described assays may be used to identify compounds capable of attenuating or inhibiting the rate or extent of cellular adhesion.

ICAM-1 is a cell surface glycoprotein expressed on non-hematopoietic cells such as vascular endothelial cells, thymic epithelial cells, certain other epithelial cells, and fibroblasts, and on hematopoietic cells such as tissue macrophages, mitogen-stimulated T lymphocyte blasts, and germinal centered S cells and dendritic cells in tonsils, lymph nodes, and Peyer's patches. ICAM-1 is highly expressed on vascular endothelial cells in T cell areas in lymph nodes ana tonsils showing reactive hyperplasia. ICAM-1 is expressed in low amounts on peripheral blood lymphocytes. Phorbol ester-stimulated A S 3 - 01. W P 021789 2 4 " 8 differentiation of some myelomonocytic cell lines greatly increases I CAM-1 expression. Thjs, ICAM-1 is preferentially expressed at sites of inflammation, and is not generally expressed by quiescent cells. ICAM-1 expression on dermal fibroblasts is increased threefold to fivefold by either interleukin 1 or gamma interferon at levels of 10 U/ml over a period of 4 or 10 hours, respectively. The induction is dependent on protein and mRNA synthesis and is reversible.

ICAM-1 displays molecular weight heterogeneity in different cell types with a molecular weight of 97 kd on fibroblasts, 114 kd on the myelomonocytic cell line U937, and 90 kd on the B lymphoblastoid cell JY. ICAM-1 biosynthesis has been found to involve an approximately 73 kd intracellular precursor. The non-N-glycosylated form resulting from tunicamycin treatment (which inhibits clycosylation) has a molecular weight of 55 kd.

ICAM-1 isolated from phorbol ester stimulated US37 cells or from fibroblast cells yields an identical major product having a molecular weight of 60 kd after chemical deglycosylation. ICAM-1 monoclonal antibodies interfere with the adhesion of phytohemacalutinin blasts to LFA-1 deficient cell lines. Pretreatment of fibroblasts, but not lymphocytes, with monoclonal antibodies capable cf binding ICAM-1 inhibits lymphocyte-fibroolast adhesion. Pretreatment of lymphocytes, but not fibroblasts, with antibodies against LFA-1 has also been found to inhibit lymphocyte-fibrobl ast adhesion.

ICAM-1 is, thus, the binding ligand of the CO' 18 complex on leukocytes. It is inducible on fibroblasts and endothelial cells j_n vitro by inflammatory mediators such as IL-1, gamma interferon and tumor necrosis factor in a time frame consistent with the infiltration of lymphocytes into inflammatory lesions in vivo (Dustin, M.L., et^. al.. J. Immunol 137:245-254. (1936); Prober. J.$., et. al.. J. Immunol 137:1893-1896, (1986)). Further ICAM-1 is expressed on non-hematopoietic cells such as vascular endothelial cells, thymic epithelial ceils, other epithelial cells, and fibroblasts and on hematopoietic cells such as tissue macopnages, mitogen-stimulated T A33-01.WP 031789 2 44 3 5 lymphocyte blasts, and germinal center 3-cells and dendritic cells in tonsils, lymph nodes and Peyer's patches (Oustin, M.L., et. si.. vL I-munol ]32:2i5-254, (1SS5)). ICAM-1 is expressed on keratinocytes in benign inflammatory lesions such as allergic eczema, lichen planus, exanthema, urticaria and bullous diseases. Allergic skin reactions provoked by the application of a hasten on the skin to which the patient is allergic also revealed a heavy ICAM-1 expression on the keratinocytes. On the other hand toxic patches on the skin did not reveal ICAM-1 expression on the keratinocytes. ICAM-1 is present on keratinocytes from biopsies of skin lesions from various dermatological disorders and ICAM-1 expression is induced on lesions from allergic patch tests while keratinocytes from toxic patch test lesions failed to express ICAM-1.

ICAM-1 is, therefore, a cellular substrate to which ly-pnocyt.es can attach, so that the lymphocytes may migrate to sites cf inflammation and/or carry out various effector functions contributing to this inflammation. Such functions include the production of antibody, lysis of virally infected target cells, etc. The term "inflammation," as used herein, is meant to include reactions of the specific snd-ncr. specific defense systems. As used herein, the term "specific defense system" is intended to refer to that component cf the immune system that reacts to the presence of specific antigens. I n 7 ! c" ation is said to result from a response of the specific defense system if the inflammation is caused by, mediated by, or associated' with a reaction of the specific defense system. Examples of inflammation resulting from a response of the specific defense system include the response to antigens such as rubella virus, autoimmune diseases, delayed type hypersensitivity response mediated by T-cells (as seen, for example in individuals who test "positive" in the Ma.ntaux test), psoriasis, etc.

A53-01.W? C 317 89 2 4 4 8 5 5 A 'non-specific de:en se sys tem reaction" is a response mediated by leukocytes incapable of immunological memory. Such calls induce granulocytes and macrophages. As usee herein, inflammation is said to result from a response of the non-specific defense system, if the inflammation is caused by, mediated by, or associated with a reaction of the non-specific defense system. Examples of inflammation which result, at least in part, from a reaction of the non-specific defense system include inflammation associated with conditions such as: asthma; acult respiratory distress syndrome CARDS) or multiple organ injury syndromes secondary to septicemia or trauma; reperfusior. injury of myocardial or other tissues; acute glomerulonephritis; reactive arthritis; dermatoses with acute inflammatory components; acute purulent meningitis or other central nervous system inflammatory disorders; thermal injury; hemodialysis; leukapheresis ; ulcerative colitis; Crohn 's disease; nectrot i z i r.c enterocolitis, granulocyte transfusion associated syndromes; and cytokine induced toxicity.

In accordance with the present invention, ICAM-1 functional derivatives, and especially such derivatives which comprise fragments or mutant variants of ICAM-1 which possess domains 1, 2 and 3 can be used in the treatment or therapy of such reactions of the non-specific defense system. More preferred for such treatment or therapy are ICAM-1 fragments or mutant variants which contain domain 2 of ICAM-1. Most preferred for such treatment or therapy are ICAM-1 fragments or mutant variants which contain domain 1 of ICAM-1.

C. Cloning of the ICAM-1 Gene Any of a variety of procedures may be used to clone the ICAM-1 gene. One such method entails analyzing a shuttle vector library of cQNA inserts (derived from an ICAM-1 axoressing cell) for the presence of an insert which contains the ICAM-1 gene. Such an analysis may be conducted by transfecting ceils with the vector and then assaying for ICAM-1 expression. The preferred method for cloning this gene entails determining the ammo acid sequence of the ICAM-1 molecule. To accsmonsn "his tasx ICAM-I protein may be ourified anc analyzes by 2 448 5 automated saquenators. Alternatively, the molecule may be fragmented as with cyanogen bromide, or with proteases sucr. as papain, chymotrypsin or trypsin (Oike, Y. et al.. J. Biol. C'ne^. 257 :9751 -9753 (1982); Liu, C. et al.. Int. J. Peot. Protein Res. 21:209-215 ( 1983 )). Although it is possible to determine the entire amino acid sequence of ICAM-1, it is preferable to determine the sequence of peotide fragments of the molecule. If the peptides are greater than 10 amino acids long, the sequence information is generally sufficient to permit one to clone a gene such as the gene for ICAM-1.

The sequence of amino acid residues in a peptide is desianatec herein either through the use of their commonly employed 3-letter designations or by their single-letter designations. A listing of these 3-letter and 1-letter designations may be found in textbooks such as Biochemistrv. Lenninger, A., Worth Publishers, New York, NY (1970). When such a sequence is listed vertically, the amino terminal residue is intended to be at the top of the list, and the carboxy terminal residue of the peptide is intended to be at the bottom of the list. Similarly, when listed horizontally, the amino terminus is intended to be on the left end whereas the carboxy terminus is intended to be at the right end. The residues of amino acids in a peptide may be separated by hyphens. Such hyphens are intended solely to facilitate the presentation of a sequence. As a purely illustrative example, the amino acid sequence designated: -Gly-Ala-Ser-Phe- indicates that an Ala residue is linked to the carboxy group of Gly, and that a Ser residue is linked to the carboxy group of the Ala residue and to the amino group of a Phe residue. The designation further indicates that the amino acid sequence contains the tetrapeptide Gly-Ala-Ser-Phe. The designation is not intended to limit the amino acid sequence to this one tetraoeptide, but is intended to include (1) the tetrapeptide having one or more amine acid residue: linked to either its amino or carboxy end, (2) the tetrapeptide having one or more amino acid residues linked Ho both its amino anc its A93-01.WP G2 2^48 carboxy encs, (3) the tetrapeptide having no additional ami no acid residues.

Once one or more suitable peptide fragments have been sequenced, the CNA sequences capable of encoding then are examined. Because the genetic code is degenerate, more than one codon may be used to encode a ^articular amino acid (Watson, J.D., In: Molecular 3iolocv of the Gene. 3rc Ed., W.A. Benjamin, Inc., Menlo Park, CA ( 1977), pp. 356-357). The peptide fragments are analyzed to identify sequences of amino acids which may be encoded by oligonucleotides having tne lowest cegree of degeneracy. This is preferably accomplished by identifying sequences that contain amino acids which are encoded by only a single codon. Although occasionally such amino acid sequences may be encoded by only a single oligonucleotide, frequently the amino acid sequence can be encoded by any of a set of similar oligonucleotides. Importantly, whereas all of the members of the set contain oligonucleotides which are capable of encoding the peptide fragment and, thus, potentially contain the same nucleotide sequence as the gene which encodes the peptide fragment, only one member of the set contains a nucleotide sequence that is identical to the nucleotide sequence of this gene. Because this member is present within the set, and is capable of hybridizing to DNA even in the presence of the other members cf the sat, it is possible to employ the unfractionatsd set of oligonucleotides- in the same manner inShich one would employ a single oligonucleotide to clone the gene that encodes the peptide.

In a manner exactly analogous to that described above, one may emDloy an oligonucleotide (or set of oligonucleotides) which have a nucleotide sequence that is complementary to the oligonucleotide sequence or set of sequences that is caoable of encoding the peptide fragment.

A suitable oligonucleotide, or set of oligonucleotides which is capable of encoding a fragment of the ICAM-1 gene (or which is complementary to such an oligonucleotide, or set of oligonucleotides) is identified (using the above-describec procedure), synthesized, anc -93-01 .UP 031789 2 4 4 8 5 3 hyoridized, by means well known in the arc, against a CNA or, ir.cre preferably, a cDNA preparation derived from human cells which are capable of expressing ICAM-1 gene sequences. Techniques of nucleic acid hybridization are disclosed by Maniatis, I. et a;.. In: Molecular Cloning, a Laboratory Manual. Coldspring Harbor, NY (1982), anc by Haymes, 3.0. et al.., In: Nucleic Acid Hvbriza'Jcn. a Practical Approach. IRL Press, Washington, 0C (1985), which references are herein incorporated by reference. The source of ONA or cCNA used will preferably have been enriched for ICAM-1 sequences. Such enrichment can most easily be obtained from cDNA obtained by extracting RNA from cells culturec under conditions which induce ICAM-1 synthesis (such as US37 grown in the presence of phorbol esters, etc.).

Techniques such as, or similar to, those described above have successfully enabled the cloning of genes for human aldenyce dehydrogenases (Hsu, L.C. et al., Proc. Natl. Acad. Sci. USA 82:3771-3775 (1985)), fibronectin (Suzuki, S. et al.. Eur. MoT . Biol. Organ. J. £:2519-2524 (1935)), the human estrogen receptor gene (Walter, P. et al ., Proc. Natl. Acad. Sci. USA 82:7889-7893 (1935)), tissue-type plasminogen activator (Pennica, 0. et al.. Nature 301:21^-221 (1983)) and human term placental alkaline phosphatase complementary DNA (Kam, W. et al., Prcc. Natl. Acad. Sci. USA 82:3715-3719 ( 1935)).

In a preferred alternative way of cloning the ICAM-1 gene, a library of expression vectors is prepared by Cloning ONA or, more preferably cQNA, from a cell capable of expressing ICAM-1 into an expression vector. The library is then screened for members capaole of expressing a protein which binds to anti-ICAM-1 antibody, and which has a nucleotide sequence that is capable of encoding polypeptides that nave the same amino acid sequence as ICAM-1 or fragments cf ICAM-1.

The cloned ICAM-1 gene, obtained through the methods described above, may be operably linked to an expression vector, and introduced into bacterial, or eukaryotic cells to produce iCAM-I protein. Techniques for such manipulations are disclosed by Mar.iatis, T. et al..

S'jpra. and are well known in the art.

A33-01.W? 03:739 2 448 5 3 D. Uses of Assays of LFA-1 Dependent Accusation The above-described assay, capable of measuring LFA-1 dependent aggregation, .nay be employed to identify agents which act as antagonists to inhibit the extent of LFA-1 dependent aggregation. Such antagonists may act by impairing the ability of LFA-1 or of ICAM-1 to mediate aggregation. Thus, such agents include immunoglobulins such as an antibody capable of binding to either LFA-1 or I CAM-i. Additionally, non-immunoglobul in (i.e., chemical) agents may be examined, using the above-described assay, to determine whether they are antagonists of LFA-1 aggregation.

E. Uses of Antibodies Capable of Binding to ICAM-1 Recestor Proteins 1. Anti-1nflammatory Agents Monoclonal antibodies to members of the CD 18 complex inhibit many adhesion dependent functions of leukocytes including binding to endothelium (Haskard, D., et al.. J. Immunol. 137-.2901-2906 (1986)), homotypic adhesions (Rothlein, R., et al.. J. Exo. Med. 153:1 132-1 149 (19S6)), antigen and mitogen induced proliferation of lymphocytes (Davignon, D., et al .. Proc. Natl.s Acad. Sci.. USA 78:4535-4539 (1S81)), antibody formation (Fischer, A., et al.. J. Immunol. 136:3198-3203 ( 1986)), and effector functions of all leukocytes such as lytic activity of cytotoxic T cells (Krensky, A.M., et al .. J. Immunol . 132:2180-2182 (1984)), macrophages (Strassman, G., et al.. J. Immunol . 136:4328-4333 (1986)), and all cells involved in antibody-dependent cellular cytotoxicity reactions (Kohl, S., et al.. J. Immunol. 113:2972-2978 (1984)). In all of the above functions, the antibocies inhibit the ability of the leukocyte to adhere to the appropriate cellular substrate which in turn inhibits the final outcome.

A93-0I .WP 031789 2 4 4 P r 3 As discussed above, the binding of ICAM-1 molecules to the members of LFA-1 family of molecules is of central importance in cellular adhesion. Through the process of adhesion, lymphocytes are capable of continually monitoring an animal for the presence of foreign antigens. Although these processes are normally desirable, they are also the cause of organ transplant rejection, tissue graft rejection and many autoimmune diseases. Kence, any means capable of attenuating or inhibiting cellular adhesion would be highly desirable in recipients of organ transplants, tissue grafts or autoimmune patients.

Monoclonal antibodies capable of binding to ICAM-1 are highly suitable as anti-inflammatory agents in a mammalian subject. Significantly, such agents differ from general anti-inflammatory agents in that they are capable of selectively inhibiting adhesion, and do not offer other side effects such as nephrotoxicity which are found with conventional agents. Monoclonal antibodies capable of binding to ICAM-1 can therefore be used to prevent organ or tissue rejection, or modify autoimmune responses without the fear of such side effects, in the mammalian subject.

Importantly, the use of monoclonal antibodies capable of recognizing ICAM-1 may permit one to perform organ transplants even between individuals having HLA mismatch. 2. Suppressors of Delayed Type Hypersensitivity Reaction Since ICAM-1 molecules are expressed mostly at sites of inflammation, such as those sites involved in delayed type hypersensitivity reaction, antibodies (especially monoclonal antibodies) capable of binding to ICAM-1 molecules have therapeutic potential in the attenuation or elimination of such reactions. This potential therapeutic use may be exploited in either of two manners.

First, a composition containing a monoclonal antibody against ICAM-1 may be ac.r.i ni stered to a patient experiencing delayed type hypersensitivity reaction. For example, such compositions might be oroviced A93-01.WP 031723 2 4 B 5 to a individual who had been in contact with antigens such as poison ivy, poison oak, etc. In the second embodiment, the monoclonal antibody capable of binding to ICAM-1 is administered to a patient in conjunction with an antigen in order to prevent a subsequent inflammatory reaction. Thus, the additional administration of an antigen with an ICAM-l-binding monoclonal antibody may temporarily tolerize an individual to subsequent presentation of that antigen. 3. Therapy for Chronic Inflammatory Oisease Since LAD patients that lack LFA-1 do not mount an inflammatory response, it is believed that antagonism of LFA-l's natural ligand, ICAM-1, will also inhibit an inflammatory response. The ability of antibodies against ICAM-1 to inhibit inflammation provides the basis for their therapeutic use in the treatment of chronic inflammatory diseases and autoimmune diseases such as lupus erythematosus, autoimmune thyroiditis, experimental allergic encephalomyelitis (EAE), multiple sclerosis, some forms of diabetes Reynaud's syndrome, rheumatoid arthritis, etc. Such antibodies may also be employed as a therapy in the treatment of psoriasis. In general, tne monoclonal antibodies capable of binding to ICAM-1 may be employed in the treatment of those diseases currently treatable through steroid therapy. s 4. Diagnostic and Prognostic Applications Since ICAM-1 is expressed mostly at sites of inflammation, monoclonal antibodies capable of binding to ICAM-1 may be employed as a means of imaging or visualizing the sites of infection and inflammation in a patient. In such a use, the monoclonal antibodies are detectably labeled, through the use of radioisotopes, affinity labels (such as biotin, avidin, etc.) fluorescent labels, paramagnetic atoms, etc. Procedures for accomplishing such labeling are well known to the art. -93-01 .WP 031739 2 <M '= 5 3 Clinical apolication of antibodies in diagnostic imaging are reviewed by Grossman, H.B., Urol. CI in. North Amer. JJ3:4C5-474 (1986)), Unger, E.C. et al., Invest. Radiol. 20:693-700 ( 19S5)), and Khaw, S.A. et al.. Science 209:295-297 (1980)).

The presence of inflammation may also be detected through the use of binding ligands, such as mRNA, cDNA, or DNA which bind to ICAM-1 gene sequences, or to ICAM-1 mRNA sequences, of cells which express ICAM-1. Techniques for performing such hybridization assays are described by Maniatais, T. [suora).

The detection of foci of such detectably labeled antibodies is indicative of a site of inflammation or tumor development. In one embodiment, this examination for inflammation is done by removing samples of tissue or blood and incubating such samples in the presence of the detectably labeled antibodies. In a preferred embodiment, this technique is done in a non-invasive manner through the use of magnetic imaging, fluorography, etc. Such a diagnostic test may be employed in monitoring organ transplant recipients for early signs of potential tissue rejection. Such assays may also be conducted in efforts to determine an individual's predilection to rheumatoid arthritis or other chronic inflammatory diseases.

. Adjunct to the Introduction of Antigenic Material Administered for Therapeutic or Diagnostic Purposes Immune responses to therapeutic or diagnostic agents such as, for example, bovine insulin, interferon, tissue-type plasminogen activator or murine monoclonal antibodies substantially impair the therapeutic or diagnostic value of such agents, and can, in fact, causes diseases such as serum sickness. Such a situation can be remedied through the use of the antibodies of the present invention. In this embodiment, such antibodies would be administered in combination with the therapeutic or diagnostic agent. The addition of the antibodies prevents the recipient from recognizing the agent, and therefore prevents the A93-01 .WP 031739 recipient from initiating an immune response against it. The absence of such an immune response results in the ability of the patient to receive additional administrations of the therapeutic or diagnostic agent.

F. Uses of Intercellular -dhesion Moleculs-l (ICAM-I) ICAM-I is a binding partner of LFA-I. As such, ICAM-1 or its functional derivatives may be employed interchangeably with antibodies capable of binding to LFA-1 in the treatment of disease. Thus, in solubilized form, such molecules may be employed to inhibit inflammation, organ rejection, graft rejection, etc. ICAM-1, or its functional derivatives may be used in the same manner as anti-ICAM antibodies to decrease the immunogenicity of therapeutic or diagnostic agents.

ICAM-1, its functional derivatives, and its antagonists may be used to block the metastasis or proliferation of tumor cells which express either ICAM-1 or LFA-1 on their surfaces. A variety of methods may be used to accomplish such a coal. For example, the migration of hematopoietic calls requires LFA-1-ICAM-1 binding. Antagonists of such binding therefore suppress this aigration and block the metastasis of tumor cells of leukocyte lineage. Alternatively, toxin-derivatized molecules, capable of binding either ICAM-1 or a member of the LFA-1 family of molecules, may be administered to a patient. When such toxin-derivatized molecules bind to tumor cells that express ICAM-1 or a member of the LFA-1 family of molecules, the presence of the toxin kills the tumor cell thereby inhibiting the proliferation of the tumor.

G. Uses of Non-Immunoqlooulin Antaconists of ICAM-I Dependent Adhesion ICAM-1-dependent adhesion can be inhibited by non-immunoglobulin antagonists which are caaable of binding .to either ICAM-1 or to LFA-1. One example of a non-immunoglobulin antagonist of ICAM-1 is LFA-1. An example of a non-immuncglobul in antagonist which binds to LFA-1 is AS3-01.WP 031783 £ h 4 R ^ ICAM-1. Through the use of the above-described assays, additional non-immunoglobul in antagonists can be identified anc purified. Non-immunoglobul i n antagonists of ICAM-1 dependent adhesion may be used for the same purpose as antibodies to LFA-1 or antibodies to ICAM-1.

H. Admin• strstion of the Compositions of the Present Invention The therapeutic effects of ICAM-1 may be obtained by providing to a patient the entire ICAM-1 molecule, or any therapeutically active peptide fragments thereof.

ICAM-1 and its functional derivatives may be obtained either synthetically, through the use of recombinant DNA technology, or by proteolysis. The therapeutic advantages of ICAM-1 may be augmented through the use of functional derivatives of ICAM-1 possessing additional amino acid residues added to enhance coupling to carrier or to enhance the activity of the ICAM-1. The scope of the present invention is further intended to include functional derivatives of ICAM-1 which lack certain amino acid residues, or which contain altered amino acid residues, so long as such derivatives exhibit the capacity to affect cellular adhesion.

Both the antibodies of the present invention and the ICAM-1 molecule disclosed herein are said to be "substantially free of natural contaminants" if preparations which contain them are substantially free of materials with which these products are normally and naturally found.

The present invention extends to antibodies, and biologically active fragments thereof, (whether polyclonal or monoclonal) which are capable of binding to ICAM-1. Such antibodies may be produced either by an animal, or by tissue culture, or recombinant DNA means.

In providing a patient with antibodies, or fragments thereof, capable cf binding to ICAM-1, or when providing [CAM-i (or a fragment, variant, cr derivative thereof) to a recipient patient, the dosage of administered agent will vary depending upon such factors as the A93-01.W? 031739 € 4 ■': & 5 3 oatient's age, weight, height, sex, general medical condition, previous medical history, etc. In general, it is desirable to provide tr.e recipient with a dosage of antibody which is in the range of from about 1 pg/kg to 10 mg/kg (body weight of patient), although a lower cr higher dosage may be administered. When providing ICAM-1 molecules cr their functional derivatives to a patient, it is preferable tc administer such molecules in a dosage which also ranges from about 1 og/kg to 10 mg/kg (body weight of patient) although a lower or higher dGsage may also be administered. As discussed below, the therapeutically effective dose can be lowered if the anti-ICAM-1 antibody is additionally administered with an anti-LFA-1 antibody. As used herein, one compound is said to be additionally administered witn a second compound when the administration of the two ccmpouncs is ir. such proximity of time that both compounds can be detected at the sa~e time in the patient's serum.

Both the antibody capable of binding to ICAM-1 and ICAM-1 itself may be administered to patients intravenously, intramuscularly, subcutaneously, enterally, or parenterally. When administering antibody or ICAM-1 by injection, the administration may be By continuous infusion, or by single or multiple boluses.

The anti-inflammatory agents of the present invention are intencec to be provided to recipient subjects in an amount sufficient to suppress inflammation. An amount ^s said to be sufficient to "suppress" inflammation if the dosage, route of administration, etc. cf the agent are sufficient to attenuate or prevent inflammation.

Anti-ICAM-1 antibody, or a fragment thereof, may be administsrsc either alone or in combination with one or more additional immunosuppressive agents (especially to a recipient of an organ c-tissue transplant). The administration of such compound(s) may be for either a "prophylactic" or "therapeutic" purpose. When proviceo prophylactical1y, the immunosuppressive compound(s) are provided in advance of any inflammatory response or symptom (for examcle, prior to. at, or shortly after) the time of an organ or tissue transoiant but : AS3-01 .WP 2 4^85 3 advance of any symptoms of organ rejection). The prophylactic administration of the compound(s) serves to prevent or attenuate any subsequent inflammatory response (such as, for example, rejection of a transplanted organ or tissue, etc.). When provided therapeutically, the immunosuppressive camoound(s) is provided at (or shortly after) the onset of a symptom of actual inflammation (such as, for example, organ or tissue rejection). The therapeutic administration of the compound(s) serves to attenuate any actual inflammation (such as, for examole, the rejection of a transplanted organ or tissue).

The anti•inflammatory agents of the present invention may, thus, be provided either prior to the onset of inflammation (so as to suppress an anticipated inflammation) or after the initiation of inflammation.

A comoosition is said to be "pharmacologically acceptable" if its administration can be tolerated by a recipient patient. Such an agent is said to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant. An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient.

The antibody and ICAM-1 molecules of the present invention can be formulated according to known aethods to prepare pharmaceutically useful compositions, whereby these materials, or t.neir functional derivatives, are combined in admixture with a pharmaceutically acceptable carrier vehicle. Suitable 'vehicles and their formulation, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in Remington's Pharmaceutical Sciences (15th sc., Qsoi, A., Ed., Hack, Easton PA ( 1930)). [n order to form a pharmaceutical!;/ acceptable composition suitable for effective administration, such compositions will contain an effective amount of anti-ICAM antibody or ICAM-1 molecule, or their functional derivatives, together with a suitable amount of carrier vehicle.

Additional pharmaceutical methods may be employed to control the duration of action. Control release preparations may be achieved through the use of polymers to complex or absorb ant'-ICAM-1 antibocy A53-OI.WP 03 1739 i 44 8 5 ? or I CAM-1, or tneir functional derivatives. The controlled delivery ,-nay be exercised by selecting appropriate macromoiecules (for example :olyesters, polysmino acids, polyvinyl, pyrrolidone, ethylenevinyl -acetate, methyl cellulose, carboxymethylcellulose, or protamine, sulfate) and the concentration of macromolecules as well as the methods of incorporation in order to control release. Another possible method to control the duration of action by controlled release preparations is to incorporate anti-ICAM-i antibody or ICAM-1 molecules, or their functional derivatives, into particles of a polymeric material such as polyesters, polyamino acics, hydrogels, poly(lactic acid) or ethylene vinyl acetate copolymers. Alternatively, instead of incorporating these agents into polymeric particles, it is possible to entrap these materials in microcapsules prepared, for example, by coacervation techniques or by ir.terfacial polymerization, for example, hydroxymethylcellulose or gelatins-microcapsules and poly-(methylmethacylate) microcapsules, respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (19S0).

Having now generally described the invention, the same will be more readily understood throucr, reference to the following examples which are provided by way of illustration^ and are not intended to be limiting of the present invention, unless specified.

EXAMPLE 1 Cultaring of Mammalian Cells l In general, the E3V-transformed and hybridoma cells of the present invention were maintainec in RMPI 16*0 culture medium, supplemented with 20 mM L-glutamine, 50 ng/ml gentamicin, and 10% fetal bovine (or fetal calf) sera. Cells were cultured at 37*C in a 5« CO?, 95% humidity atmosohere. -32-01.'«? 031739 To establish Epstein-3arr virus (E3V) transformants, 10° T cell depleted peripheral blood mononuclear cells/ml in RPMI 1640 medium supplemented with 20% fetal calf serum (FCS), and 50 pg/ml gentamicin were incubated for 15 hours with EBV-containinc supernatant of B95-3 cells (Thorley-Lawson, D.A. et aj_., J. Exoer. Med. ii6:4S5 (1977)).

Cells in 0.Z ml aliquot were placed in 10 microtiter wells. Medium was replaced with RPMI 1640 medium (supplemented with 20% fetal calf serum and 50 ^c/~: gentamicin) until cell growth was noted. Cells grew in .tost wells and were expanded in the same medium. Phytonemagglutinin (PHA) blasts were established at 10° cells/ml in RPMI 1640 medium (supplemented with 20% fetal calf serum) containing a 1:800 dilution of PHA-P (Difco Laboratories, Inc., Detroit, MI). PHA lines were expanded with interleukin 2 (IL-2)-conditioned medium ana pulsed weekly with PHA (Cantrell, D.A. et al.. J. Exper. Med. 158:1895 (1983)). The above procedure was disclosed by Springer, T. et al., J. Exoer. Med. 160:1901-1913 (1984), which reference is herein incorporated by reference. Cells obtained through the above procedure are then screened with anti-LFA-1 antibodies to determine whether they express the LFA-1 antigen. Such antibodies are disclosed by Sanchez-Madrid, F. et al., J. Exoer. Med. 158:1785 (1983).

EXAMPLE 2 Assays of Cellular Aggregation and Adhesion In order to assess the extent of cellular adhesion, aggregation assays were employed. Cell lines used in such assays were washed two times with RPMI 1640 medium containing 5 mM Hepes buffer (Sigma Chemical Co., St. Louis) and resuspended to a concentration of 2 x 10° cells/ml. Added to flat-bottomed, 96-well microtiter plates (No. 3596; Costar, Cambridge, MA) were 50 /il of appropriate monoclonal antibody suoernaUnt or 50 p.] of complete medium with or without purified monoclonal antibodies, 50 ^1 of complete medium containing 200 ng/rnl of the phorbol ester phorbol myristate acetate (PMA) and 100 ^1 of cells AS3-01.WP 031729 244,; at a concentration of 2 x 10° cells/ml in complete medium. This yielde.1 a final concentration of 50 ng/ml PHA and 2 x 1(P cells/well. Cells were allowed to settle spontaneously, and the degree of aggregation was scored at various time points. Scores ranged from 0 to 5-r, where 0 indicated that essentially no cells were in clusters; 1-r indicated that less than 10% of the cells were in aggregates; 2+ indicated that less than 50% of the cells were aggregated; 3r indicated that up to 100% of the cells -were in-small, loose clusters; 4-indicated that up to 100% of the cells were aggregated in larger clusters; and 5r indicated that 100% of the cells were in large, very compact aggregates. In order to obtain a more quantitative estimate of cellular adhesion, reagents and cells were added to 5 ml polystyrene tubes in the same order as above. Tubes were placed in a rack on a gyratory shaker at 37*C. After 1 hour at approximately 200 rpm, 10 /il of the cell suspension was placed in a hemocytometer and the number of free cells was quantitated. Percent aggregation was determined by the following equation: number of free cells % aggregation = 100 x (1 ) number of input cells The number of input cells in the above formula is the number of cells per ml in a control tube containing onliy cells and complete n.edium that had not been incubated. The number of free cells in the above equation equals the number of non-aggregated cells per ml from experimental tubes. The above procedures were described by Rothlein, R., et al .. vh Exoer. Med. 153:1132-1149 (1936).

EXAMPLE 3 LFA-1 Dependent Cellular- Aggregation The qualitative aggregation assay described in Example 2 was performed using the 'Epstein-Barr transfanrred 'ceil Vine JV. Upon A92-01 .WP 031733 2 A 4 r' addition of PMA to the culture medium in the microtiter plates, aggregation of cells was observed. Time lapse video recordings showed that the JY ceils on the bottom of the microtiter wells were motile and exhibited active membrane ruffling and pseudopodia movement. Contact between the pseudopodia of neighboring cells often resulted in cell-cell adherence. If adherence was sustained, the region of cell contact moved to the uropod. Contact could be maintained despite vigorous cell movements and the tugging of the cells in opposite directions. The primary difference between PMA-treated and untreated cells appeared to be in the stability of these contacts once they were formed. With PMA, clusters of cells developed., growing in size as additional cells adhered at their periphery.

As a second means of measuring adhesion, the quantitative assay described in Example 2 was used. Cell suspensions were shaken at 200 rpm for 2 hours, transferred to a hemocytometer, and cells not in aggregates were enumerated. In the absence of PMA, 42% (SO = 20%, N = 6) of JY cells were in aggregates after 2 hours, while JY cells incubated under identical conditions with 50 ng/ml of PMA had 87% (SO = 8%, N = 6) of the cells in aggregates. Kinetic studies of aggregation showed that PMA enhanced the rate and magnitude of aggregation at all time points tested (Figure 3).

EXAMPLE \ Inhibition of Aggregation of Cells Using Anti-LFA-1 Monoclonal Antibodies To examine the effects of anti-LFA-1 monoclonal antibodies on PMA-induced cellular aggregation, such antibodies were added to cells incubated in accordance with the qualitative aggregation assay cf Example 2. The monoclonal antibodies were found to inhibit the formation of aggregates of cells either in the presence or absence cf PMA. Both the F(ab')2 and Fab' fragments of monoclonal antibodies against the alpha chain of LFA-1 were cacable of inhibiting cellular A93-01,WP 0317=5 2 4 4 3 5 aggregation. Whereas essentially 10C% of cells formed aggregates in the absence of anti-LFA-1 antibody, less than 20% of the cells were found to be in aggregates when antibody was added. The results of this experiment were described by Rothlein, R. et aj.. (J. Exoer. Med. 153:1 132-1 H9 (1986).

EXAMPLE 5 Cellular Aggregation Requires the LFA-1 Receptor E3V-transfcrmed 1 ymphobl as to i d cells were prepared from patients in the manner described in Example 1. Such cells were screened against monoclonal antibodies capable of recognizing LFA-1 and the cells were found to be LFA-1 deficient.

The qualitative aggregation assay described in Example 2 was employed, using the LFA-1 deficient cells described above. Such cells failed to spontaneously aggregate, even in the presence of PMA.

EXAMPLE o The Discovery of ICAM-1 The LFA-1 deficient calls of Example 5 were labeled with carboxyfluorescein diacetate (Patarroyp, M. et al., Cel1. Immunol. £3:237-248 (1981)). ' The labeled cells were mixed in.a ratio of 1:10 with autologous or JY cells and the percentage of fluorescein-! abeled cells in aggregates was determined according to the procedure of Rothlein, R. et al.. J. Exoer. Med. 163:1132-1149 ( 1986). The LFA-1 deficient cells were found to be capable of coaggregating with LFA-1 expressing cells (Figure 4).

To determine whether LFA-1 was important only in forming aggregates, or in their maintenance, antibodies capable of binding to LFA-1 were added to the preformed aggregates described above. The addition of antibody was found to strongly disrupt the preformed aggregation. Time A93-01.WP 031739 2 4 4 - 3S - lapse video recording confirmed that addition of the monoclonal antibodies to preformed aggregates began to cause disruption within 2 hours (Table 1). After addition of monoclonal antibodies against LFA-1, pseudopodial movements and changes in shape of individual cells within aggregates continued unchanged. Individual cells gradually disassociatec from the periphery of the aggregate; by 8 hours ceils were mostly dispersed. By video time lapse, the disruption of preformed aggregates by LFA-1 monoclonal antibodies appeared equivalent to the aggregation process in the absence of LFA-1 monoclonal antibody running backwards in time.

TA3LE 1 Ability of Anti-LFA-1 Monoclonal Antibodies to Disrupt Preformed PMA-Induced JY Cell Aggregates Exp.

Acoreaation score 2 ha 18 h -mAb rinAb 1 4+ 4r l*b 2 3r 4r 1+C 3 + + v 1-d Aggregation in the qualitative microtiter plate assay was scored visually. With anti-LFA-1 present throughout the assay period, aggregation was less than 1+. aAmount of aggregation just before addition of Mono-lonal antibody at 2 h. bTS1/IS - TS1/22.

CTS1/18. dTS1/22.

A93-01.WP 021739 2 44 v EXAMPLE 7 The Requirement of Divalent Ions for LFA-1 Dependent Aggregation LFA-1 dependent adhesions between cytotoxic T cells and targets require the presence of magnesium (Martz, E. J. Cell. 3io1. 34:534-593 (1930)). PMA-induced JY cell aggregation was tested for divalent cation dependence. JY cells failed to aggregate (using the assay c~ Example 2) in medium free of calcium or magnesium ions. The addition of divalent magnesium supported aggregation at concentrations as low as 0.3 mM. Addition of calcium ions alone had little effect. Calcium ions, however, were found to augment the ability of magnesium ions to support PMA-induced aggregation. When 1.Z5 mM calcium ions were added to the medium, magnesium ion concentrations as low as 0.02 millirno1 ar were found to support aggregation. These data show that the LFA-i dependent aggregation of cells requires magnesium ions, and that calcium ions, though insufficient of themselves, can synergize with magnesium ions to permit aggregation.

EXAMPLE 8 The Isolation of Hybridoma Cells Capable of Expressing Anti-ICAM-1 Monoclonal Antibodies Monoclonal antibodies capable of binding to ICAM-1 were isolated according to the method of Rothlein, R. et al.. J. Immunol . 137:1270-1274 (1986), which reference has been incorporated by reference herein. Thus, 3 BAL8/C mice were immunized intraperi toneal ly with E27-transformed peripheral blood mononuclear cells from an LFA-l-deficient individual (Springer, T.A. et al.., J. Exoer. Med. 160:1901 (1984)).

Approximately IO'7 cells in 1 ml RPMI 1540 medium was used for each immunization. The immunizations were administered 45, 29, and 4 days before spleen cells were removed from the mice in order to produce tr.e A92-01.WP 031 2 4 4$ desired hyoridona cell lines. On day 3 before the removal of the spleen cells, the mice were given an additional 10' cells in 0.15 ml medium (intravenously).

Isolated ssleen cells from the above-described animals were fused with P3X73Ac3.553 myeloma cells at a ratio of 4:1 according to the protocol of Galfre, G. et al.. Nature 266:550 (1977). Aliquots of the resulting hybridoma cells were introduced into 96-well microtiter plates. The hybridoma suoernatants were screened for inhibition of aggregation, and one inhibitory hybridoma (of over 500 wells tested) was clonec and subclonec by limiting dilution. This subclone was designated RRL/1.1.1 (hereinafter designated "RR1/1").

Monoclonal antibody RR1/1 was consistently founc to inhibit PMA-stimulated aggregation of the LFA-1 expressing cell line JY. The RR1/1 monoclonal antibody inhibited aggregation equivalently, or slightly less than seme monoclonal antibodies to the LFA-1 alpha or beta subunits. In contrast, control monoclonal antibody against HLA, which is abundantly expressed on JY cells, did not inhibit aggregation. The antigen bound by monoclonal antibody RR1/1 is defined as the intercellular adhesion molecule-1 (ICAM-1).

EXAMPLE 9 Use of Anti-ICAM-1 Monoclonal Antibodies to Characterize the ICAtt-1 Molecule In order to determine the nature of ICAM-1, and particularly to determine whether ICAM-1 was distinct from LFA-1, cell proteins were immunoprecipitated using monoclonal antibocy RR1/1. The immunoprecipitation was performed according to the method of Rothlein, R. et al . (J. Immunol. J_3_7:1270-1274. (1986)). JY cells were lysed at 5 x 10^ cells/ml in \% Triton X-100, 0.14 m NaCl, 10 mM Tris, pH 8.0, with freshly added 1 mM phenylmethylsulfanylfluoride. 0.2 units per ml trypsin innibitor aprotinin (lysis buffer) for 20 minutes at 4'C. Lysates were centrifuged at 10,000 x g for 10 minutes and precleared A93-01.WP 021739 C v f ' y» L 4 ^ o 0 with 50 of a 50% suspension of CNBr-activated, glycine-quenched Sepharose C1--3 for 1 hour at 4*C. One milliliter of lysate was immunoprecipitated with 20 /xl of a 50% suspension of monoclonal antibody RRI/1 coupled to Sepharose CI-48 (I tng/ml) overnight at 4*C (Springer, T.A. et al., J. Exoer. Med. ,150: 1901 (1984)). Seprtarose- bound monoclonal antibody was prepared using CNBr-activation of Sepharose CL-43 in carbonate buffer according to the method of March, S. et a].. (Anal. Biochem. 60:149 (1974)). Washed immunoprecipitates were subjected to SDS-PAGE and silver staining according to the procedure of Morrissey, J.H. Anal. Biochem. 117:307 ( 1981).

After elution of proteins with SDS sample buffer (Ho, M.K. et al.. J. Biol. Chetn. 258:536 ( 1983)) at 100'C, the samples were divided in half anc subjected to electrophoresis (SDS-8% PAGE) under reducing (Figure :A) or nonreducing conditions (Figure 53). Bands having molecular weights of 50 kd and 25 kd corresponded to the heavy and light chains of immunoglobulins from the monoclonal antibody Sepharose (Figure 5A, lane 3). Variable amounts of other bands in the 25-50 kd weight range were also observed, but were not seen in precipitates from hairy leukemia cells, which yielded only a 90 kd molecular weight band. The 177 kd alpha subunit and 95 kd beta subunit of LFA-1 were found to migrate differently from ICAM-1 under Doth reducing (Figure 5A, lane 2) and nonreducing (Figure 5B, lane 2) conditions.

In order to determine the effect of monoclonal antibody RRI/1 on PHA-lympnoblast aggregation, the quantitative aggregation assay described in Example 2 was employed. Thus, T cell blast cells were stimulated for 4 days with PHA, thoroughly washed, then cultured for 5 da, in the presence of IL-2 conditioned medium. PHA was found to be internalized during this 6-day culture, and did not contribute to the aggregation assay. In three different assays with different T cell blast preparations. ICAM-1 monoclonal antibodies consistently inhibiced aggregation (Table 2).

A93-01,W= 031789 2 4 4 B 5 TABLE 2 Inhibition of PMA-Stimulated PHA-Lymphobiast Aggregation by RRI/1 Monoclonal Antibody a/ ot /© /o Exot. PMA MAb Aggregation Inhibition'3 Conirol 9 Control 51 0 HLA-A,3 53 -14d LFA-1 alpha 31 39 ICAM-1 31 39 Control Control 78 0 LFA-1 beta 17 78 ICAM-1 50 — 7 Control 70 HLA-A,B 80 -14 LFA-3 83 -19 LFA-1 alpha 2 97 LFA-1 beta 3 96 ICAM-1 34 51 Aggregation of PHA-induced lymphoblasts stimulated with 50 ng/ml PMA was auantitated indirectly by microscopically counting the number of nonagcregaled cells as described in Example 2.

^Percent inhibition relative to 'tells treated with PMA and X63 monoclonal antibody.- Aggregation was measured 1 hr after the simultaneous addition of monoclonal antibody and PMA. Cells were shaken at 175 rpm.

^A negative number indicates percent enhancement of aggregation.

Aggregation was measured 1 hr after the simultaneous addition of monoclonal antibody and PMA. Cells were pelleted at 200 x G for 1 min. incubated at 37*C for 15 min. gently resuspended, and shaken for 45 min. at 100 rpm. rCe11s were pretreated with PMA for 4 hr at 37*C. After monoclonal antibody was added, the tubes were incubated at 37*C stationary for 20 min. and shaken at 75 rpm for 100 min.

A84.1.W? 09213S ) /. /-. a L 4 -j LFA-1 monoclonal antibodies were consistently more inhibitory than ICAM-1 monoclonal antibodies, whereas HLA-A, B and LFA-3 monoclonal antibodies were without effect. These results ■ indicate that of the monoclonal antibodies tested, only those capable of binding to LFA-1 or ICAM-1 were capable of inhibiting cellular adhesion.

EXAMPLE 10 Preparation of Monoclonal Antibody to ICAM-I I~mun ization A Salb/C mouse was immunized intraperitoneally (i.p.) with 0.5 mis of 2 x 10-' JY cells in RPMI medium 103 days and 24 days prior to fusion. On day 4 and 3 prior to fusion, mice were immunized i.p. with 1G' cells of PMA differentiated U937 cells in 0.5 ml of RPMI medium.

Differentiation of U937 Cells U937 cells (ATCC CRL-1593) were differentiated by incubating them at 5 x lOVml in RPMI with 10" Fetal Bovine Serum, 1% glutamine and 50 ^a/ml centamyin (complete medium) containing 2 ng/ml phorbol-12-myristate acetate (PMA) in a sterile polypropylene container. On the third day of this incubation, one-half of tfie volume of medium was withdrawn and replaced with fresh complete medium containing PMA. On day 4, cells were removed, washed and prepared for immunization.

Fusion Spleen cells from the immunized mice were fused with P3x63 Ag8-553 myeloma cells at a 4:1 ratio according to Gal fre et a]_., (Nature 265:550 (1977)). After the fusion, cells were plated in a 96 well flat bottomed microtiter plates at 10^ spleen cells/well.

Selection for Anti-ICAM-I Positive Cells After one week, 50 p] of supernatant were screened in tne qualitative aggregation assay of Example 2 using both JY and SKVI-3 as aggregating cell lines. Cells from supernatants inhibiting JY cell aggregation but not SKW-3 were selected and cloned 2 times utilizing 1imi t i ng dilution. 2 4 4 3 This experiment resulted in the identification and cloning of three separate hybridoma lines which proc'uced anti - [CAM-1 monoclonal antibodies. The antibodies produced by these hybridoma lines were Ig^2a» and respectively. The hybridoma cell line which produced the IgG^a anti-ICAM-1 antibody was given the designation R6 '5'06 'E9 '52. The antibody produced by the preferred hybridoma cell line was designated R6'5'Do 'ES '62 (herein referred to as "R6-5-D6").

EXAMPLE 11 The Expression and Regulation of ICAM-1 In order to measure ICAM-1 expression, a raaioimmune assay was developed. In this assay, purified RRI/1 was ioainated using iodogcr. to a specific activity of 10 iiCi/^g. Endothelial cells were grown in 96 well plates and treated as described for each experiment. The plates were cooled at 4*C by placing in a cold room for 0.5-1 hr, not immediately on ice. The monolayers were washed 3x with cold complete media and tnen incubated 30 m at 4'C with ^5j rri/i. The monolayers were then washed 3x with complete media. The bound ^5j was released using 0.1 N NaOH and counted. The specific activity of the ^'-1 RRI/1 was adjusted using unlabeled RRI/1 to obtain a linear signal over the range of antigen densities encountered in this study. Non-specific binding was determined in the presence of a thousand fold excess of unlabeled RR1/1 and was subtracted from total binding to yield the speci fic binding.

ICAM-1 expression, measured using the above described radioimmune assay, is increased on human umbilical vein endothelial cells (HUVEC) and human saphenous vein endothelial cells (HSVEC) by IL-1, TNF, LPS and I FN garrma (Table 3). Saphenous vein endothelial cells were used in this study to confirm the results from umbilical vein endothelial cells in culture*: large vein endothelial cells derived from adult tissue. The basal expression of ICAM-1 is 2 fold higher on saphenous vein endothelial cells than on umbi1ical vein encothel ial cells. Exposure 2 4;; 3 5 3 of umbilical vein endothelial cell to recombinant IL-1 alpha, IL-1 beta, and INF gamma increase ICAM-1 expression 10-20 fold. IL-I alpha. INF and LPS were the most potent inducers and IL-1 was less potent on a weight basis and also at saturating concentrations for the response (Table 3). IL-1 beta at 100 ng/ml increased ICAM-1 expression by 9 fold on HUVEC ana 7.3 fid on HSVEC with naif-maximal increase occuring at 15 ng/ml. rTNF at 50 ng/ml increased ICAM-1 expression 16 fold on HUVEC and 11 fold on HSVEC with half maximal effects at 0.5 ng/ml. interferon-gamma produced a significant increase in ICAM-1 expression of 5.2 fold on HUVEC or 3.5 fold on HSVEC at 10,000 U/'ml. The effect of LPS at 10 Mg/ml was similar in magnitude to that of rTNF. Pairwise combinations of these mediators resulted in acditive or slightly less than additive effects on ICAM-I expression (Table 3). Cross-titration of rTNF with rIL-1 beta or rI FN gamma showed no synergism between these at suboptimal or optimal concentrations.

Since LPS increased ICAM-1 expression on endothelial cells at levels sometimes found in culture media, the possibility that the basal ICAM-I expression might be due to LPS was examined. When several serum batchs were tested it was found that low endotoxin serum gave lower ICAM-1 basal expression by 25%. All the results reported here were for endothelial cells grown in low endotoxin serum. However, inclusion of the LPS neutralizing antibiotic polymyxin B at 10 /xg/ml decreased ICAM-1 expression only an additional 25% (Table 3). The increase in ICAM-1 expression on treatment with IL-1 or TNF was not effected by the presence of 10 (xg/ml polymyxin B which is consistent with the low endotoxin levels in these preparations (Table 3). 2 4 4853 - 4/ TABLE 3 Anti-ICAM-1 Monoclonal Antibodies Condition (15 hr) control 100 ng/ml rIL-1 beta 50 ng/ml rIL-1 alpha 50 ng/ml rTNF alpha 10 /ig/ml LPS 10 ng/ml rI FN gamma rIL-I beta + rTNF rIL-1 beta + LPS rIL-1 beta + rIFN gamma rTNF + LPS rTNF + rIFN gamma LPS + I FN gamma polymyxin B (10 /ig/ml) polymyxin B -1- rIL-1 polymyxin B + rTNF 1 /ig/ml LPS polymyxin B ♦ LPS 125i Specifi HUVEC 603 * 11 5580 + 633 9x 9910 ± 538 15x 9550 + 1500 16x 9530 + 512 16x 3120 ± 303 .2x 1459 + 1410 24x 13986 + 761 23x 7849 + 601 13x 15364 + 1241 24x 13480 + 1189 22x 10206 + 320 17x 430 - 23 • 5390 - 97 llx 9735 * 389 20x 75S8 + 432 13x 510 + 44 l.lx 11y bound (CPM) HSVEC 1132 * 31 8320 ± 765 7.3x 12690 ± 657 11.2x 10459 + 388 S. 2:< 4002 ±~664 3.5x 15269 + 660 14x 10870 + 805 lOx 8401 * 390 7.4x 16141 + 1272 14x 13238 + 751 12x 10987 + 668 lOx Upreaulation of tCAM-l expression on HVEC ifld HSVEC- HUVEC or HSVEC were seeded into 96 well plates at 1:3 from a confluent monolayer and allowed to grow to confluence. Cells were then treated with the indicated materials or media for 15 hr and the RIA done as in methods. All points were done in quadruplicate. s 2 4 4 EXAMPLE 12 Kinetics of Interleukin 1 and Gamma Interferon Induction of ICAM-1 The kinetics of interleukin 1 and gamma interferon effects on ICAM-1 expression on dermal fibroblasts were determined using the ^--1 goat anti-mouse IcG binding assay of Dustin, M.L. et a],. (J. Immunol.

J_3_7: 2^5 - 25-i (1S86); which reference is herein incorporated by reference). To perform this binding assay, human dermal fibroblasts were grown in a 96-well microtiter plate to a density of 2-S x 10^ cells/well (0.32 cm-). The cells were washed twice with RPMI 1640 medium suco 1 emented as described in Example 1. The cells were additionally washed once with Hanks Balanced Salt Solution (H3SS), 10 mM HEPES, 0.05% NaNj and 10% heat-inactivated fetal bovine serum. Washing with this binding buffer was done at 4'C. To each well was added 50 /:! of the above-described binding buffer and 50 p.] of the appropriate hybridoma supernatant with X63 and W6/32 as the negative and positive controls, respectively. After incubation for 30 minutes at 4'C, with gentle agitation, the wells were washed twice with binding buffer, and the second antibody l^I-goat anti-mouse IgG, was added at 50 nCi in 100 /xl. The ^3I-goat anti-mouse antibccy was prepared by using lodocen (Pierce) according to the method of Fraker, P.J. et al. (Biochem. Bioohvs. Res. Commun. 80:84^ (1978)). After 30 minutes at a*C, the wells were washed twice with 200 p.] of binding buffer and the cell layer was solubilized by adding 100 p'\ of 0.1 N NaCH. This and a 100 p] wash were counted in a Beckman 5500 gamma counter. The specific counts per minute bound was calculated as [cpm with monoclonal antibody]-[cpm with X63]. All steps, including induction with specific reagents, were carried out in quadruplicate.

The effect of interleukin 1 with a half-life for ICAM-l induction of 2 hours was more rapid than that of gax.ma interferon with a half-life of 3.75 hours (Figure 6). The time course of return to resting levels of ICAM-1 appeared to depend upon the cell cycle or rate of cell growth. In cuiescent cells, interleukin 1 and gamma interferon effects are stable for 2-3 days, whereas in log phase cultures, ICAM-1 o /, a n expression is near baseline 2 days after the removal of these inducing agents.

The dose response curves for induction of ICAM-1 by recombinant mouse and human interleukin 1, and for recombinant human gamma interferon, are shown in Figure 7. Gamma interferon anc interleukin 1 were found to have similar concentration dependencies with nearly identical effects at 1 ng/~l . The human and incuse recombinant interleukin 1 also have similar curves, but are much less effective than human interleukin 1 preparations in inducing I CAM-i expression.

Cyclohexamiae, an inhibitor of protein synthesis, and actincmycin 0, an inhibitor of mRNA synthesis, abolish the effects cf both interleukin 1 and gamma interferon on ICAM-1 expression on fibroblasts (Table 4). Furthermore, tunicamycin, an inhibitor of N-linkea glycosylation, only inhibited the interleukin 1 effect by 43%. These results indicate that protein and mRNA synthesis, but not N-linked glycosylation, are required for interleukin 1 and gamma interferon-stimulated increases in ICAM-1 expression.

V, 24*85 50 - TABLE - Effects of Cycloheximide, Actinomycin D, anc Tunicamycin on ICAM-1 Induction by IL 1 anc gamma I FN on-Human Dermal- Fibroblasts3 *^I Goal Anti-Mouse IcG Specifically Bound fcpm! "^eatment anti -ICAM-1 ant: -HLA-A,g.C Control (- hr) 1524 ± 140 11923 - 5CC • cycloheximice 1513 7 10 10573 - 471 •r actinomycin D 1590 45 12275 A 60S tunicamycin 1451 - 176 12340 - 9-0 IL 1 (10 U/ml) (4 hr) 4254 249 12155 510 •r cycloheximice 1519 + 3S1 12675 ± 445 ■r actinomycin 0 1513 + 88 12294 r 123 -r tunicamycin 30S4 4. 113 13434 — 661 IFN-y (10 U/ml) (13 hr) 4659 109 23675 — 500 + cycloheximide 1451 59 10675 ± 300 + actinomycin D 1325 ± 186 12089 r 550 aHuman fibroblasts were grown to'a density of S x IO4 cells/0.32 cm^ well. Treatments were carried out in a final volume of 50 ji) containing the indicated reagents. Cycloheximide, actinomycin D, and tunicamycin were addec- at 20 ^g/ml, 10 uM, and 2 ^g/mi, respectively, at the same time as the cytokines. All points are means of quadruplicate wells z SD. v.

EXAMPLE 13 The Tissue Distribution of ICAM-1 Histocr.emical studies were performed on frozen tissue of human organs to determine the distribution of ICAM-1 in thymus, lymph nodes, intestine, skin, kidney, and liver. To perform such an analysis, frozen tissue sections (4 iim thick) of normal human tissues were fixed in acetone for 10 minutes and stained with the monoclonal antibody, RRI/1 by an immunoperoxidase technique which employed the avidin-biotin complex method (Vector Laboratories, Surlingame, CA) described by Cerf-3ensussar,, N. et al . fj. Immunol . 12 C: 2515 ( 1933 )). After incubation with the antipccy, tr.e sections w ere sequentially incubated with bi ot i nyi ated nor:e anti-mouse IgG a r,z avid in-biot my! ated pe^oxicase complexes. The sections were finally dipped in a solution containing 3-amino-9-etuyl-carbazole (Aldrich Chemical Co., Inc., Milwaukee, WI) to develop a color reaction. The sections were then fixed in 4% formaldehyde for 5 minutes and "were counterstained with hematoxylin. Controls included sections incubated with unrelated monoclonal antibodies instead of the RRI/1 antibody.

ICAM-1 was found to have a distribution most similar to that of the major histccompatibil ity complex (MHC) Class II antigens. Most of the blood vessels (both small and large) in all tissues showed staining of endothelial cells with ICAM-1 antibody. The vascular endothelial staining was more intense in the interfollicular (paracortical) areas in lymph nodes, tonsils, and Payer's patches as compared with vessels in kidney, liver, and normal skin. In the liver, the staining was mostly restricted to sinusoidal lining cells; the hepatocytes and the endothelial cells lining most of the portal veins and arteries were not stained.

In the thymic medulla, diffuse staining of large cells and a dendritic staining pattern was observed. In the cortex, the staining pattern was focal and predominantly dendritic. Thymocytes were not stained. In the peripheral lymphoid tissue, the germinal center cells of the secondary lymphoid follicles were intensely stained. In some lymphoid follicles, the staining pattern was mostly dendritic, with no recognizable staining of lymphocytes. sFaint staining of cells in the mantle zone was also observed. In addition, dendritic cells with cytoplasmic extensions (interdigitating reticulum cells) and a small number of lymphocytes in the interfoll icular or paracortical areas stained with the ICAM-1 binding antibody.

Cells resembling macrophages were stained in the lymph nodes anc! lamina prooria of the small intestine. Fibroblast-1ike cells (spindle-shaped cells) and dendritic cells scattered in the stroma of most of the organs studied stained with the ICAM-1 binding antibody. No staining was discerned in the Langerhans/indeterminant cells in the epidermis. No staining was observed in smooth muscle tissue.

The staining of epithelial cells was consistently seen in the mucosa of the tonsils. Althougn hepatocytes, bile duct epithelium, intestinal eoit'nelia' cells, and tubular eoithelial cells in kidney did not stain 2 h ') B 5 3 ir. most circumstances, sections of normal kidney tissue obtained from a necrectomy specimen with renal cell carcinoma showed staining of many proximal tubular cells for ICAM-1. These tubular epithelial cells also stained with an anti-HLA-DR binding antibody.

In summary, ICAM-1 is expressed on non-hematopoietic cells such as vascular encothelial cells and on hematopoietic cells such as tissue macrophages ana mitogen-stimul ated I lymphocyte blasts. ICAM-1 was found to be expressed in low amounts on peripheral blood lymphocytes.

EXAMPLE 14 The Purification of ICAM-1 by Monoclonal Antibody Affinity Chromatography General purification scheme ICAM-1 was purified from human cells or tissue using monoclonal antibody affinity chromatography. Monoclonal antibody, RRI/1T reactive with ICAM-1 was first purified, and coupled to an inert column matrix. This antibody is described by Rothlein, R. et al. J. Immunol . 137:1270-1274 (1986), and Oustin, M.L. et al. (0. Immunol. 137:245 (1585). ICAM-1 was solubilized from cell membranes by losing the cells in a ncn-ionic detergent, Triton X-100, at a near neutral p'H. The cell lysate containing solubilized ICAM-1 was then passed through pre-cDiumns designed to.remove materials £hat bind nonspecifically to the column matrix material, and then through the monoclonal antibody column matrix, allowing the ICAM-1 to bind to the antibody. The antibody column was then washed with a series of detergent wash buffers of increasing pH uo to pH 11.0. Ouring these washes ICAM-1 remained bound to the antibody matrix, while non-binding and weakly binding contaminants were removed. The bound ICAM-1 was then specifically eluted from tne column by applying a detergent buffer of pH 12.5. i f i cat i on of monoclonal antibody RR1/1 and covalent coupling to Seoha^ose CL--S.

The ant'-'.CAM-l monoclonal antibody RRI/1 was purified from the i::: tes of hybridoma-bearing mice, or from hybridoma culture : tec standard techmcues of ammonium sulfate precipitation and ?. 4 A 8 5 3 protein A affinity chromatography (Ey et a].., Immunochem. 15:429 (1973)). The purified IgG, or rat IgG (Sigma Chemical Co., St. Louis, MO) was covalently coupled to Sepharose CL-4B (Pharmacia, Upsala, Sweden) using a modification of the method of March et ah (Anal.

Bioche^. 50:149 (1974)). Briefly, Sepharose CL--8 was washed in distilled water, activated with <10 mg/ml CNBr in 5 M KjHPQ^ (pH approximately 12) for 5 minutes, and then washed extensively with 0.1 mM HC1 at 4*C. The filtered, activated Sepharose was resuspended with an eaual volume of purified antibody (2-10 mg/ml in 0.1 M NaHCOj, 0.1 M NaCl). The suspension was incubated for 18 hours at 4*C with gentle end-over-end rotation. The supernatant was then monitored for unbound antibody by absorbance at 280 nm, and remaining reactive sites on the activated Sepharose were saturated by adding glycine to 0.05 M. Coupling efficiency was usually greater than 90%.

Oetercent solubilization of membranes prepared from human soleen.

All procedures were done at 4'C. Frozen human spleen (200 g fragments) from patients with hairy cell leukemia were thawed on ice in 2C0 ml Tris-saline (50 mM Tris, 0.14 M NaCl, pH 7.4 at 4*C) containing 1 mM phenylmethyl sulfonyl fluoride (PMSF), 0.2 U/ml aprotinin, and 5 mM iodoacetamide. The tissue was cut into small pieces, and homogenized at 4*C with a Tekmar power homogenizer. The volume was then brought to 300 ml with Tris-saline, and 100 ml of 10% Tween 40 (polyoxyethylene sorbitan monopalmitate) in Tris-saline was added to achieve a final 1 concentration of 2.5% Tween 40.

To prepare membranes, the homogenate was extracted using three strokes of a Dounce or, more preferably, a Teflon Potter Elvej'nem homogenizer, and then centrifuged at 1000 x g for 15 minutes. The supernatant was retained and the pellet was re-extracted with 200 ml of 2.5% Tween <*o in Tris-saline. After centrifugation at 1 GOO x a for i5 minutes, the supernatants from both extractions were combined and centrifuged at 150,000 x g for 1 hour to pellet the membranes. The membranes were washed by resusper.ding in 200 ml Tris-saline, centr:fuged at 150,000 x g for i hour. The membrane pellet was -e3u::ended in 200 ml Tris-saline ana was homogenized with a motorizes i zer and Teflon pestle until the suspension «as uniformly 2 4 4 8 5 3 turbid. The volume was then brought up to 900 ml with Tris-saline, and N-lauroyl sarcosine was added to a final concentration cf 1%. After stirring at 4*C for 30 minutes, insoluble material in the detergent 1ysate was removed by centrifugation at 150,000 x g for 1 hour. Triton X-100 was then added to the supernatant to a final concentration of 2%, and the lysate was stirred at 4"C for 1 hour.

Ceteraent solubilization of JY 8-1vmonoblastoid cells The EBV-transformed B-lymphoblastoid cell line JY was grown in RPMI -1540 containing 10% fetal calf serum (FCS) and 10 mM KEPES to an approximate density of 0.3 - 1.0 x 10° cells/ml. To increase the cell surface expression of ICAM-1, phorbol 12-myristate 13-acetate (PMA) was added at 25 ng/ml for 8-12 hours before harvesting the cells. Sodium vanadate (50 /:M) was also added to the cultures during this time. The cells were pelleted by centrifugation at 500 x g for 10 minutes, and washed twice in Hank's Balanced Salt Solution (HBSS) by resuspension and centrifugation. The cells (approximately 5 g per 5 liters of culture) were lysed in 50 ml of lysis buffer (0.14 M NaCl, 50 mM Tris pH 8.0, 1% Triton X-1C0, 0.2 U/ml aprotinin, 1 mM PMSF, 50 sodium vanadate) by stirring at 4*C for 30 minutes. Unlysed nuclei and insoluble debris were removed by centrifugation at 10,000 x g for 15 minutes, followed by centrifugation of the supernatant at 150,000 x g for 1 hour, and filtration of the supernatant through Whatman 3mm filter paper.

Affinity chromatography of ICAM-1 for structural studies For large scale purification of ICAM-1 to be used in structural studies, a column of 10 ml of RR1/1-Sepharose Cl-43 (coupled at 2.5 mg of antibody/ml of gel), and two 10 ml pre-columns of CNBr-activated, glycine-quenched Sepharose CL-43, and rat-IaG coupled to Sepharose CL-48 (2mg/ml) were used. The columns were connected in series, and pre-washed with 10 column volumes of lysis buffer, 10 column volumes of pK 12.5 buffer (50 mM triethylamine, 0.1% Triton X-100, pH 12.5 at 4*C), followed by equilibration with 10 column volumes of lysis buffer. One liter of the detergent lysate of human spleen was loaced at a flow rate of 0.5-1.0 ml per minute. The two pre-columns were uceo to remove non- Ih specifically binding material from the lysate before passage through the RRl/l-Sepnarose column.

After loading, the column of RRl/i-Sepharose and bound ICAM-1 was washed sequentially at a flow rate of i 'ml/minute with a minimum of 5 column volumes each of the following: 1) lysis buffer, 2) 20 mM Tris pH 8.0/0.14 M NaCl/0.1% Triton X-100, 3) 20 mM glycine pH 10.0/0.1% Triton X-100, and 4) 50 mM tri ethyl amine pH 11.0/0.1% Triton X-100. All wash buffers contained 1 mM PMSF and 0.2 U/ml aprotinin. After washing, the remaining bound ICAM-1 was elutac with 5 column volumes of elution buffer (50 mM triethyl amine/0.1% Triton X-100/pH 12.5 at 4'C) at a flow rate of 1 ml/3 minutes. The eluted ICAM-I was collected in 1 ml fractions and immediately neutralized by the addition of 0.1 ml of 1 M Tris, pH 6.7. Fractions containing ICAM-1 were identified by SDS-polyacrylamide electrophoresis of 10 /il aliquots (Springer et al., jh Exp. Med. 150:1901 (1984)), followed by silver staining (Morrissey, J.H., Anal. Biochem. 117:307 (1981)). Under these-conditions, the bulk of the ICAM-1 eluted in approximately 1 column volume and was greater than 90% pure as judged from silver-stained electropherograms (a small amount of IgG, leeched from the affinity matrix, was the major contaminant). The fractions containing ICAM-1 were pooled and concentrated approximately 20-fold using Centricon-30 microconcentrators (Amicon, Danvers, MA). The purified ICAM-1 was quantitated by Lowry protein assay ofsan ethanol-precipitated aliquot of the pool: approximately 500 fig of pure ICAM-1 were produced from the 200 g of human spleen.

Approximately 200 (iq of purified ICAM-1 was subjected to a second stage purification by preparative SDS-pol yacryl amide gel electrophoresis. The band representing ICAM-1 was visualized by soaking the gel in 1 M KC1. The gel region which contained ICAM-1 was then excised and electroeluted according to the method of Hunkapiller et al_., Meth. Enzvmol. £1:227 -236 ( 1933). The purified protein was greater than 98% pure as judged by SDS-PAGE and silver staining.

Affinity purification of ICAM-1 for f-jncnonal stucies ICAM-1 for use in functional stjcies was purifies from detergent lysates of JY cells as descries above, -ut on a smallscale (a 1 mi £•4853 - DO * column of RRl/l-Sepnarose), and with the following modifications. Ail solutions contained 50 sodium vanadate. After washi.ig the column with pH 11.0 buffer containing 0.1% Triton X-100, the column was washed again with five column volumes of the same buffer containing 1% n-octyl-beta-0-glucopyranoside (octylglucoside) in place of 0.1% Triton X-100. The octylglucoside detergent displaces the Triton X-100 bound to the ICAM-1, and unlike Triton X-100, can be subsequently removes by dialysis. The ICAM-1 was then eluted'with pH 12.5 buffer containing if, octylglucoside in place of 0.1% Triton X-100, and was analyzes and concentrated as described above.

EXAMPLE 15 Characteristics of Purified ICAM-1 ICAM-1 purified from human spleen migrates in SOS-polyacrylamide gels as a broad band of Mr of 72,000 to 91,000. ICAM-1 purified from JY cells also migrates as a broad band of Mr of 75,500 to 97,000. These Mr are within the reported range for ICAM-1 immunoprecipitated from different cell sources: Mr=90,000 for JY calls, 114,000 cn the myelomonocytic cell line U937, and 97,000 on fibroblasts (Dustin et al-, J. Immunol. ]_37:245 (1986)). This wide range in Mr has beer, attributed to an extensive, but variable degree of glycosylation. The non-glycosyl ated precursor has a Mr oY 55,000 (Dustin et al .). The protein purified from either JY cells or human spleen retains its antigenic activity as evidenced by its ability to re-bind to the original affinity column, and by immunoprecipi tation with SRi/'l-Sepharose and SOS-polyacrylamide electrophoresis.

To produce peptide fragments of ICAM-1, approximately 200 iiq was reduced with 2 mM dithiothreitol/2% SOS, followed by alkylation with 5 mM iodoacetic acid. The protein was precipitated with ethanoi. anc redissolved in 0.1 M NH4CO3/O.1 mM CaCl2/0.1% zwittergent 3-1-(Calbiochem), and digested with 1% w/w trypsin at 37*C for <1 hours, followed by an additional digestion with 1% trypsin for 12 hours a*. 37"C. The tryptic peptides were purified by reverse-phase HPLC using a 0.4 x 15 cm C- column (Vydac). The peptides were elutes witn a linear Gradient or 0% to 50% acetonitrile in 0.1% tn f luoroacetis as is. 2 4 4 8 5 Selected peptides were subjected to sequence analysis on a gas phase microsequenator (Applied BiosysU.ns). The sequence information obtained from this study is shown in Tab!s 5.

TABLE 5 Ami no Acid Sequences of ICAM-1 Trvotic Peptides Amino Peptide Acid Residue 50a 5Gb 4oa 45b X 45 K ~A J U Q Ml ] [T/V] A (V/A) E V S 1 L. £ 4 n L V L 2 r r S Q P E F N 1 G L T L/E 3 L T T A L P P D S G L P/(G) 4 T s F A A T i_ V I P V L P P P V 3 L E G/Y y G L L N T p V T N/L 7 P W P P V V I Q T P (N) 8 i P I I T/I G G C P/V (Q) S s F G (G) L - L S K (E) £ E (Q) - 0 r" L.

T (0) 11 A S 0/P K s i_ S 12 G/S V V P r r F C 13 A T D Q s 0 K G V W V/L A - Q I K T P S K 17 A 13 P 19 s V A 0 21 L ( ) = Low confidence sequence.

[ ] = Very low confidence sequence.

/ = Indicates ambiguity in the sequence; most probable amino acid is listed first. a ® Major peptide. b = Minor peptide.

I k B b EXAMPLE 15 Cloning of the ICAM-1 Gene The gene for ICAM-1 may be cloned using any of a variety of procedures. For example, the amino acid sequence information obtained through the sequencing of the tryptic fragments of ICAM-I (Table 5) can be used to identify an oligonucleotide sequence which would correspond to the ICAM-1 gene. Alternatively, the ICAM-1 gene can be cloned using anti-ICAM-1 antibody to detect clones which produce ICAM-1.

Cloning of the aene for ICAM-1 through the use of oligonucleotide probes Using the genetic code (Watson, J.O., I_n: Molecular Sioloav of the Gene. 3rd Ed., 11.A. Benjamin, Inc., Menlo Park, CA (1977)), one or more different oligonucleotides can be identified, each of which would be capable of encoding the ICAM-1 tryptic peotides. The probability that a particular oligonucleotide will, in fact, constitute the actual ICAM-1 encoding sequence can be estimated by considering abnormal base pairing relationships and the frequency with which a particular codon is actually used (to encode a particular amino acid) in eukaryotic cells. Such "codon usage rules" are disclosed by Lathe, R., et al.. vL MoTec. Biol . 183:1-12 (1935). Using trfe "codon usage rules" of Lathe, a single oligonucleotide, or a set of oligonucleotides, that contains a theoretical "most probable" nucleotide sequence (i.e. the nucleotide sequence having the lowest redundancy) capable of encoding the ICAM-1 tryptic peptide sequences is identified.

The oligonucleotide, or set of oligonucleotides, containing the theoretical "most probable" sequence caoable of encoding the ICAM-1 fragments is used to identify the sequence of a cc~.pl err.entary oligonucleotide or set of oligonucleotides which is capable of hybridizing to the "most probably" sequence, or set of sequences. An oligonucleotide containing such a comolementary sequence can be employed as a probe to identify and isolate the ICAM-1 gene (Mar.iatis. T., et al., Molecular Cloning A Laboratory Manual. Cols Serine -arscr Press, Colo Soring Harbor, NY (19321. 24'95 3 - 59 As described in Section C, suora, it is possible to clone the ICAM-1 gene from eukaryotic DNA preparations suspected of containing this gene. To identify and clone the gene which encodes the ICAM-1 protein, a DNA library is screened for its ability to hybridize with the oligonucleotide probes described above. Because it is likely that there will be only two copies of the gene for ICAM-1 in a normal diploid cell, and because it is possible that the ICAM-1 gene may have large non-transcribed intervening sequences (intrcns) wr.ose cloning is not desired, it is preferable to isolate ICAM-l-ancoding sequences from a cDNA library prepared from the mRNA of an ICAM-I-producing cell, rather than from genomic DNA. Suitable DNA, or cDNA preparations are enzymatically cleaved, or randomly sheared, and liaated into recombinant vectors. The ability cf these recombinant vectors to hybridize to the above-described oligonucleotide probes is then measured. Procedures for hybridization are disclosed, for example, in Maniatis, T., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (1SS2) or in Haymes, B.T., et al.. Nucleic Acid Hybridization a Practical Approach. IRL Press, Oxford, England (1985). Vectors found capable of such hybridization are then analyzed to determine the extent and nature of the ICAM-1 sequences which they contain. Based purely on statistical considerations, a gene such as that which encodes the ICAM-1 molecule could be unambiguously identified (via hybridization screening*) using an oligonucleotide probe having only 18 nucleotides.

Thus, in summary, the actual identification of ICAM-1 peptide sequences permits the identification of a theoretical "most probable" DNA sequence, or a set of such sequences, capable of encoding such a peptide. By constructing an oligonucleotide complementary to this theoretical sequence (or by constructing a set of oligonucleotides complementary to the set of "most probable" oligonucleotides), one obtains a ONA molecule (or set of ONA molecules), capable of functioning as a probe to identify and isolate the ICAM-1 gene.

Using the ICAM-1 peptide sequences of Table 5. the sequence of tne "most probable" sequence of an oligonucleotide csoable of encoding the AA and J peptides was determines (Tables 6 anc 7, respectively). 01 iccnucleotide: comol ary to t oese ceo"encei 'k®'? synthesized an 9 k 8 5* purified for use as probes to isolate ICAM-1 gene sequences. Suitable size-selected cDNA libraries were generated from the poly(A)" RNA of both PMA-induced Hl-50 cells and from PS-stimul aiea umbilical vein endothelial cells. A size-selected cDNA library was prepared using poly(A)+ RNA from PMA-inducsd HL-60 cells according to the method of Gubler, U., et al. ((Gene 25:263-259 (1983)) and Corbi, A., et al. (EMSO J. 6:4023-4023 (1987)), which references are herein incorporated by reference.

A size-selected cDNA library was prepared using po 1 y (A J RNA from umbilical vein encothelial cells which had been stimulated for 4 hours with PS 5 ^g/ml. The RNA was extracted by homogenizing the cells in 4 M guanidinium isothiocyanate and subjecting the supernatant to ultracentrifugation through a CsCl gradient (Chirgwin, J.M., et al.. Biochem. 18:5294-5299 ( 1979)). Poly(A)"i" RNA was isolated from the mixture of total RNA species through the use of oligo (dT)-eellulose chromatography (Type 3, Collaborative Research) (Aviv, H., et al.. Proc. Natl. Acad. Sci. (USA) 69:1408-1412 (1972).

TABLE 5 Oligonucleotide Comolementary to the Most Probable Nucleotide Sequence Capable of Encoding the ICAM-1 AA Peptide Amino Acia Most ProDable Residue of ICAM-1 Sequence Encoding Complementary ICAM-1 AA Peotide AA Peotide Seauence ' -» / 2 162 Glu G C A T G C 163 Leu C G T A G C 164 Asp G C A T C G 165 Leu C G T A G C 156 Arg C G G C G C 167 Pro C G C G C G 163 Gin C G A T G C 159 Gly G C G C C G 170 Leu C s G T A .

G C 171 G1 u G C A T G C 172 Leu C G T A G C 173 Phe T A T A T A 174 Glu G C A i t 'i 353 TABLE 6 (continued) Oligonucleotide Complementary to the Most Probable Nucleotide Sequence Capable of Encoding the ICAM-1 AA Peptide Amino Acid Most Prooaoie Residue of ICAM-1 Sequence Encoding Comolementary ICAM-1 AA Peptide AA peotide Secuence 175 Asn A I A T C G 176 Thr A T C G C G 177 Ser U C A 3' 5 z ' ! 1 4 4 B 5 3 63 - TABLE 7 Oligonucleotide Complementary to the Most Probable Nucleotide Sequence Capable of Encoding the ICAM-I J Peptide Amino Acic Most Prooable Residue of ICAM-1 Sequence Encoding ComDlementary ICAM-1 AA Peotide IS Val Thr 21 Cys 22 Ser 23 Thr 24 Ser Cys 26 Asp 27 Gin 28 Pro 29 Lys Peotide Secjence ' 3' G C T A G c A t C G C G T A G r C G T A C G C G A T C G C G T A C G C G T A G C T A G C A T C s G c G A T G C C G C G c G A T A T 3' ' 09?;« O : A <" L > C First strand cDNA was synthesized using 3 pq of poiy(A)"r RNA, avian myeloblastosis virus reverse transcriptase (Life Sciences) and an oligo(dT) primer. The DNA-RNA hybrid was digested with RNase H (8RL) and the second strand was synthesizes using ONA polymerase I (New England Biolabs). The product was methylated with Eco R1 methylase (New England Biolabs), blunt end ligated to Eco R1 linkers (New England Biolabs), digested with Eco R1 and size selected on a low melting point agarose gel. cDNA greater than 500bp were ligated to XgtlO which hac previously been Eco R1 digested and depnosphorylated (Stratagene) The product of the ligation was then packaged (Stratagene gold).

The umbilical vein endothelial cell and HL-50 cDNA libraries were then plated at 20,000 PFU/15Qmm plate. Recombinant DNA was transferrec in duplicate to nitrocellulose filters, denatured in 0.5 M NaOH/l.EM NaCl, neutralized in 1M Tris, pH7.5/1.5M NaCl and baked at 80*C for 2 hours (Benton, W.D., et al., Science 195: 130-132 (1977)). Filters were prehybridizea and hybridized in 5X SSC containing 5X Dennardt's solution, 50 mM NaPO^ and 1 fig/ml salmon sperm ONA. Prehybridization was carried out at 45' for 1 hour.

Hybridization was carried out using 32i)p ('5-TTGGGCTGGTCACAG-GAGGTGGAGCAGGTGAC) or 47bp (5'-GAGGTGTTCTCAAACAGCTCCAGGCCCTGG GGCCGCAGGTCCAGCTC) anti-sense oligonucleotides based, in the manner discussed above, on the ICAM-1 tryptic peptides J and AA, respectively (Table 5 and 7) (Lathe, R.t J. Vo'lec. Biol.. 183:1-12 ( 1985)). ■j 9 Oligonucleotides were end labeled with P")ATP using T4 polynucleotide kinase and conditions recommended by the manufacturer (New England Biolabs). Following overnight hybridization the filters were washed twice with 2X SSC/0.1% SOS for 30 minutes at 45*C. Phages were isolated from those plaques which exhibited hybridization, anc were purified by successive replating and rescreening. 2 ^! Cloning of the cene for ICAM-1 through the use of anti-ICAM-1 antibody The gene for ICAM-1 may alternatively be cloned through the use of anti-ICAM-1 antibody. ONA, or more preferably cDNA, is extracted and purified from a cell which is capable of expressing ICAM-1. The purified cDNA is fragmentized (by shearing, endonuclease digestion, etc.) to prcuct a pool of DNA or cDNA fragments. DNA or cDNA fragments from this pool are then cloned into an expression vector in order to produce a genomic library of expression vectors whose members each contain a unique cloned DNA or cDNA fragment.

EXAMPLE 17 Analysis of the cDNA clones Phage CNA from positive clones were digested with Eco R1 and examined by Southern analysis using a cDNA from one clone as a probe. Maximal size cDNA inserts which cross-hybridized were subcloned into the Eco R1 site of plasmid vector pGEM 4Z (Promega). HL-60 subclones containing the cDNA in both orientations were deleted by exonuclease III digestion (Henikoff, S., Gene 28:351-359 (1984)) according to the manufacturers recommendations (Erase-a-Base, Promega). Progressively-deleted cDNAs were then cloned and subjected to dideoxynucleotide chain termination sequencing (Sanger, F. et a*l .. Proc. Natl. Acad. Sci. f USA 1 74:5463-5457 (1977)) according to the manufacturers recommendations (Sequenase, U.S. Biochemical). The HL-60 cONA 5' and coding regions were sequenced completely on both strands and the 3' region was sequenced approximately 70% on both strands. A representative endothelial cDNA was sequenced over most of its length by shotgun cloning of 4bp-recognition restriction enzyme fragments.

The cCNA sequence of one HL-60 ana one endothelial cell cDNA was established (Figure 8). The 3023 bp sequence contains a short 5' untranslated region and a 1.3 kb 3' untranslated region with a consensus colyadenylation signal at position 2965. The longest ooen reading frame begins with the first ATG at position 53 and enas with a 'I / . 4 8 TGA terminating triple: at position translated amino acid sequence and different tryptic peptides totaling figure 8) confirmed that authentic isolated. The amino acid sequences shown in Table 8. 1653. Identity between the sequences determined from 8 91 amino acids (underlines in ICAM-1 cDNA clones had been of ICAM-1 tryptic peptides are TABLE 3 Amino Acid Secuences of ICAM-1 Tryptic Peptides Peotide Residues Secuence J 14-29 X G S V L VTCSTSCDQPK U 30-39 L L G I E T ? L (P) (K) 50 78-85 (T) F L T V Y X T X 89-95 Y E L A P L ? AA 161-182 XELDLRPOGLE -- L F E X T S A P X Q L K 232-246 LNPTVTYGXDSFSAK <5 282-295 S f P A P N V (T/1) L X K P Q (V/L) Indicates that the saouence continues on the next line.

Underlined sequences were used to prepare oligonucleotide probes.

Hydrophobicity analysis (Kyte, J., et al .. J. Molec. 3io1 ., 157:105-132 (1982)) suggests the presence of a 27 residue signal sequence. The assignment of the +1 glutamine is consistent with our inability to obtain N-terminal sequence on 3 different ICAM-i protein preparations; glutamine may cyclize to pyroglumatic acid, resulting in a bloc'<e: N-terminus. The translated sequence from I to -53 is preccmi-antly hydrophilic followed by a 24 residue hydrophobic sutatwe transse-csne 24485 3 domain. The transmembrane domain is immediately followed by several charged residues contained within a 17 residue putative cytoplasmic domai n.

The predicted size of the mature polypeptide chain is 55,219 daltons, in excellent agreement with the observed size of 55,000 for deglycosyiated ICAM-1 (Dustin, M.L., et al.. J. Immunol. 137:245-254 (1986)). Eight N-linked glycosylation sites are predicted. Absence of asparagine in the tryptic peptide sequences of 2 of these sites confirm their glycosylation and their extracellular orientation. Assuming 2,500 daltons per high mannose N-linkec carbohydrate, a size of 75,000 daltons is predicted for the ICAM-1 precursor, compared to the observed six of 73,000 daltons (Dustin, M.L., et al.. J. Immunol. 137:245-254 (1986)). After conversion of high mannose to complex carbohydrate, the mature ICAM-I glycoprotein is 75 to 114 kd, depending on cell type (Dustin, M.L., et al.. J. Immunol. 137:2*5-254 (1986)). Thus ICAM-1 is a heavily glycosylated but otherwise typical integral membrane protein.

EXAMPLE 13 ICAM-1 is an Integrin-Binding Member of the Immunoglobulin Supergene family Alignment of ICAM-1 internal repeats was performed using the Microgenie protein alignment program \Queen, C., et al .. Nucl . Acid Res.. J_2:581-5 99 ( 1984)) followed by inspection. Alignment of ICAM-1 to IgM, N-CAM and MAG was carried out using Microgenie and the ALIGN program (Day'noff, M.O., et al., Meth. Enzvmol. 91:524-545 (1983)). Four protein sequence databases, maintained by the National Biomedical Research Foundation, were searched for protein sequence similarities using the FASTP program of Williams and Pearson (Lipman, D.J., et al., Science 217:1*35-1439 (1985)).

Since ICAM-1 is a ligand of an intsgrin, it was unexpected that it would be a member of the immunoglobulin supergene family. However, inspection of the ICAM-1 sequence shows that it fulfills all criteria 9 '} 5 prooosed for membership in the immunoglobulin supergene family. These criteria are dis.jussed in turn below.

The entire extracel 1 ular domain of ICAM-1 is constructed from 5 homologous i ——anocl obu 1 i n -1 i !<e domains which are shown aligned in Figure SA. Domains 1-4 are 88, 97, 99, and 99 residues long, respectively and thus are of typical Ig domain size; domain 5 is truncated wit.v.n 53 residues. Searches of the N8RF data base using the FAST? program revealed significant homologies with members of the immunoglobulin suoergene family including IgM and IgG C domains, T cell receptor a supunit variable domain, and alpha 1 beta glycoprotein (fig. S5-0).

Using the above information, the amino acid sequence of ICAM-1 was compared with the amino acid sequences of other members of the immununoglobul in supergene family.

Three types of Ig superfamily domains, V, CI, and C2 have been differentiated. 3oth V and C domains are constructed from 2 ^-sheets linked together by the intradomain disulfide bond; V domains contain 9 anti-parallel ^-strands while C domains have 7. Constant domains were divided into the CI- and C2- sets based on characteristic residues shown in Figure 9A. The Cl-set includes proteins involved in antigen recognition. The C2-set includes several Fc receptors and proteins involved in call adhesion including CD2, LFA-3, MAG, and NCAM. ICAM-1 domains were found to be most strongl>" homologous with domains of the C2 - set placing ICAM-1 in this set; this is reflected in stronger similarity to conserved residues in C2 than CI domains as shown for 3-strands B-F in Figure 9. Also, ICAM-1 domains aligned much better with 3-strands A and G of C2 domains than with these strands in V and CI domains, allowing good alignments across the entire C2 domain strength. Alignments with C2 domains from NCAM, MAG, and alpha 1-3 glycoprotein are shown in Figures 98 and 9C; identity ranged from 2S to 33".. Alignments with a T cell receptor Va 21% identity and IgM C domain 3 3<i% identity are also shown (Figures 98, 90).

One of the most imoortant characteristics of immunoglobulin domains is the di sul fide-bonded cysteines bridging the B and F 3 stra.ncs which stabilizes the 4 sheet sandwicn; in ICAM-1 the cysteines are conserved in all cases except in strand f of domain 4 where a leucine is found which may face into the sandwich and stabilize the contact as proposed for some other V- and C2-sets domains. The distance between the cysteines (43, 50, 52, anc 37 residues) is as described for the C2-set.

To test for the presence of intrachain disulfide bonds in ICAM-1, endothelial cell ICAM-1 was subjected to SOS-PAGE under reducing anc non-reducing conditions. Endothelial cell ICAM-1 was used because it shows less gIycosylation heterogeneity than JY or hairy cell splenic ICAM-1 anc allows greater sensitivity to shifts in Mr. ICAM-1 was. therefore, purified from 16 hour LPS (5 ) stimulated umbilical vein encothelial cell cultures by immunoaffinity chromatography as described above. Acetone precipitated ICAM-1 was resuspended in sample buffer (Laemmli, U.K., Mature 227:630-685 (1970)) with 0.25% 2-mer-captoethsnol or 25 mM iodoacetamide and brought to 100'C for 5 min. The samples were subjected to SOS-PAGE 4670 and silver staining 4613. Endothelial cell ICAM-1 had an apparent Mr of 100 Kd under reducing conditions and 96 Kd uncer non-reducing conditions strongly suggesting the presence of intrachain disulfides in native ICAM-1.

Use of the primary sequence to predict secondary structure (Chcu. P.Y., et al., Biochem. .13:21 1-245 (1974)) showed the 7 expected £-strands in each ICAM-1 domain, labeled a-g in Fiqure SA upper, exactly fulfilling the prediction for an immunoglobulin domain and corresponding to the positions of strands A-H in' immunoglobulins (Figure SA, lower). Domain 5 lacks the A and C strands but since these form edges of the sheets the sheets could still form, perhaps with strand 0 taking the place of strand C as proposed for some other C2 domains: and the characteristic disulfide bond between the 8 and F strands would be unaffected. Thus, the criteria for domain size, sequence homology, conserved cysteines forming the putative intradomair, disulfide bond, presence of disulfide bonds, and predicted 3 sheet structure are all met for inclusion of ICAM-1 in the immunoglobulin supergene family. £-4053 ICAM-1 was found to be most strongly homologous with the NCAM and MAG glycoproteins of the C2 set. This is of particular interest since both NCAM and MAG mediate cell-cell adhesion. NCAM is important in neuron-neuron and neuro-muscular interactions (Cunningham, 8.A., et al •, Science 216 :759-805 ( 1987)), while MAG is important in neuron-olicodendrocyte and ol igodendrocyte-oligodendrocyta interactions during myelination (Poltorak, M., et al., J. Cell Biol. 105:1893-1899 (1987)). The cell surface expression of NCAM and MAG is developmentally regulated during nervous system formation and myelination. respectively, in analogy to the regulated induction of ICAM-1 in inflammation (Springer, T.A., et al .. Ann. Rev. Immunol . 5:223-252 (1937)). ICAM-1, NCAM (Cunningham, B.A., et al.. Science 235:799-805 ( 1937)). and MAG (Salzer, J.L., et al.. J. Cell. Biol. 104:957-955 (1987)) are similar in overall structure as well as homologous, since each is an integral membrane glycoprotein constructed from 5 C2 domains forming the N-terminal extracellular region, although in NCAM some acditional r.on-Ig-like sequence is present between the last C2 domain and the transmembrane domain. ICAM-1 aligns over its entire length including the transmembrane and cytoplasmic domains with MAG with 21% identity; the same % identity is found comparing the S domains of I CAM -1 and NCAM-1. A diagrammatic comparison of the secondary structures of ICAM-1 and MAG is shown in Figure 10. Domain by domain comparisons show that the level of homology betweeh domains within the ICAM-1 and NCAM molecules (x ± s.d. 21 + 2.8% and 18.5 +3.8%, respectively) is the same as the level of homology comparing ICAM-1 domains to NCAM and MAG domains (20.4 - 3.7 and 21.9 ± 2.7, respectively). Although there is evidence for alternative splicing in the C-terminal regions of NCAM (Cunningham, 8.A., et al.. Science 235:799-806 (1987); Barthels, D., et al., EMBO J. 6:907-914 (1987)) and MAG (Lai, C., et al.. Proc. Natl. Acad. Sci. fUSA 1 84:4377-4341 ( 1987)), no evidence for this has been found in the sequencing of endothelial or HL-60 ICAM-1 clones or in studies on the ICAM-1 protein backbone and precursor in a variety of cell types (Dustin, M.L., et al.. J. Immunol. 137:245-254 ( 1986)). 4 ' * 1 L h -J ICAM-1 functions as a ligand for LFA-1 in lymphocyte interactions with a number of different cell types. Lymphocytes bind tu ICAM-1 incorporated in artificial membrane bilayers, and this requires LFA-1 on the lymphocyte, directly demonstrating LFA-1 interaction with ICAM-I (Marl in, S.D., et al .. cell £1:813-819 (1987)). LFA-1 is a leukocyte intecrin and has no immunoglobulin-1ike features. Leukocyte integrins comprise one integrin subfamily. The other two subfamilies mediate cell-matrix interactions and recognize the sequence RGQ within their ligands which induce fibronectin. vitronectin, collagen, anc fibrinogen (Hynes, R.O., CejJ. i3:5<i9-554 ( 1987); Ruoslahti, E.. et al.. Sc ience 233:491-497 ( 1987)). The leukocyte integrins are only expressed on leukocytes, are involved in cell-cell interactions, anc the only known 1 igancs are ICAM-1 and iC3b, a fragment of the complement component C3 which shows no immunoglobul in-1ike features anc is recognized by Mac-I (Kishimoto, T.K., et al.. In: Leukocyte Typing III. McMichael, M. (ed.), Springer-Verlag, New York (1937); Springer, T.A., et al. . Ann. Rev. Immunol. 5:223-252 ( 1987); Anderson, D.C., et al .. Ann. Rev. Med. 38:175-194 ( 1987)). 8ased upon secuence analysis, possible peptides within the ICAM-1 sequence recognized by LFA-1 are shown in Table 9.

TABLE S Peptides Within the I&M-I Sequence Possibly Recognized bv LFA-1 -L-R-G-E-K-E-L- -R-G-E-K-E-L-K-R-E-P- -L-R-G-E-K-E-L-K-R-E-P-A-V-G-E-P-A-E- -P-R-G-G-S- -P-G-N-N-R-K- -Q-E-D-S-Q-P-M- -T-P-E-R-V-E-L-A-P-L-P-S- -R-R-O-H-H-G-A-N-F-S- -0-L-R-P-Q-G-L-E- 2/ h, ; y .

ICAM-1 is the first example of a .nember of the immunoglobulin supergene family which binds to an intecrin. Although both of these families play an important role in cell adhesion, interaction between them had not previously been expected. In contrast, interactions within the immunoglobulin gene superfamily are quite common. It is auite possible that further examples of interactions between tne intecrin and immunoglobulin families will be found. L .r A -1 recognizes a ligand distinct from ICAM-1 (Springer, T.A., et a 1 .. A.--. Rev. Immunol. 5:223-252 (1987)), and the leukocyte intecrin Mac-1 rec:cnizes a liganG distinct from C3bi in neutrophi1-neutrophi1 adhesion (Anderson, C.C.. et al . . Ann. Rev. Med. 38: 175-194 ( 1SS7)). Furthermore, purified MAG-ccntaining vesicles bind to neurites whicn are MAG. anc thus MAG must be capable of heterophils interaction with a distinct recectcr (Poltorak, M., et al.. J. Cell Biol . 105:1833-1859 (12S7)).

NCAM's role in neural-neural and neural-muscul ar call interactions has been suggested to be due to homopnilic NCAM-NCAM interactions (Cunningham, 3.A., et al .. Science 236:755-806 (1587)). The important role of MAG in interactions between adjacent turning loops of Schwann cells enveloping axons during myelin shaath formation might be due to interaction with a distinct receptor, or due to hemophilic MAG-MAS interactions. The homology with NCAM and the frequent occurrence of domain-domain interactions within the Nirmunoglobulin supergene family raises the possibility that ICAM-I could engage in homopnilic interactions as well as I CAM-1 -LFA-1 heterophils interactions. However, binding of 8 lymphoblast cells which co-exoress similar densities of LFA-1 and ICAM-1 to ICAM-1 in artificial or cellular monolayers can be completely inhibited by pretreatment of the 3 lymphoblast with LFA-1 MAb, while adherence is unaffected by 3 lymphoblast pretreatment with ICAM-1 MAb. Pretreatment of the monolayer with ICAM-1 Mab completely abolishes binding (Dustin, M.L.. et al.. J. Immunol. 132:245-254 ( 1985); Marlin, S.D., et al.. eel' 5].:313-819 (1987)). These findings show that if ICAM-1 homuohi'ic 9 / z* L L\ 4 o interactions occur at all, they must be much weaker than heterophils interaction with Li-A-1.

The possibility that the leukocyte integrins recognize ligands in a fundamentally different way is consistent with the presence of a ISO residue sequence in their a subunits which may be important in liganc binding and which is not present in the RGO-recognizing integrins (Coroi, A., et al. fEMBQ J. c:4023--023 ( 1S87)). Although Mac-1 has been prooosed to recognize RGO sequence present in iC3b 5086, there is no RGO sequence in ICAM-1 (Fig. 8). This is i.n agreement with the failure cf the fibronectin peotice GRGOS? and the control peptide GSGES? to inhibit ICAM-1 -LFA-1 adhesion (Marl in, S.D.. et al .. eel 1 £1:313-319 (1937)). However, reiatea sequences such as PRGGS and RGEKE are present in ICAM-1 in regions predicted to loop between j} strancs a and b of domain 2 and c and d of domain 2, respectively (Fig. 9), anc thus may be accessible for recognition. It is of interest that the homologous MAG molecule contains an RGO sequence between domains 1 anc 2 (Pcltorak, M., et al.. J. Cel 1 51ol. 105:1393-1399 (1987); Salzer, J.L., et al., J. Cell. Bio'. 104:957-955 (1987)].

EXAMPLE IS Southern and Northern Blots Southern blots were performed usingsa 5 fsg of genomic DNA extracted from three cell lines: BL2, a Burkitt lymphoma cell line (a gift free. Dr. Gilbert Lenoir); JY and Er-LCL, E3V transformed S-1ymphoblas to ic cell lines.

The ONAs were digested with 5X the manufacturers recommences quantity of Bam HI and Eco R1 endonucleases (New England Biolabs). Following electrophoresis through a 0.8% agarose gel, the ONAs were transferred to a nylon membrane (Zeta Probe, 8ioRad). The filter was prehybridized and hybridized following standard procedures using I CAM cDNA from HL-60 labeled with a-(^P)d XTP's by random primir.c (Boehringer Mannheim). Northern blots were performed using 20 uc of total RNA or 6 fi g of poly(A)~ RNA. RNA was denatured ar: 2 4^8 5 3 electrophoresed through a 1% agarose-formaldehyae gel ana electrotransferreo to Zeta Probe. Filters were prehyoridized ar.d hybridized as described previously (Staunton. D.E., et al. Emoo J. 5:3695-3701 (1987)) using the HL-60 cDNA probe of ""P-labe'ied oligonucleotide probes (described above).

The Soutnern blots using the 3 kb cDNA probe anc genomic CNA digested with Sam HI and Eco R1 showed single predominant hybridizing fragments of 20 and 8 kb, respectively, suggesting a single gene anc suggesting that most of the coding information is present within S '<3. In blots of 3 different cell lines there is no evidence of restriction fragment polymorphism.

EXAMPLE 20 Expression of the ICAM-1 Gene An "expression vector" is a vector which (due to the presence of aooropriate transcriptional and/or translational control sequences) is caoable of expressing a ONA (or cDNA) molecule which has been cloned into the vector and of thereby producing a polypeptice or protein. Expression of the cloned sequences occurs when the expression vector is introduced into an appropriate host cell. If a prokaryotic expression vector is employee!*, then the appropriate host cell would be any prokaryotic cell capable of expressing the cloned sequences. Similarly, if a eukaryotic expression vector is employed, then tne appropriate host cell would be any eukaryotic eel: caoable of exoressing the cloned sequences. Importantly, since eukaryotic DNA -av contain intervening sequences, and since such sequences cannot be correctly processed in prokaryotic cells, it is preferable to employ cDNA from a cell which is capable of expressing ICAM-I in order to produce a prokaryotic genomic expression vector library. Procedures for preparing cDNA and for producing a genomic library are disclosed by Mani ati s, T., et al. (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor, NY (1932)). 9 4 4 8 5 3 KM t ^ The above-described expression vector genomic library is used to create a bank of host ceils (each of which contains one member of the library). The expression vector may be introduced into the host cell by any of a variety of means (i.e., transformation, transfecticn. protoplast fusion, electroporation, etc.). The bank of expression vector-containing cells is clonally propagated, and its members are individually assayed (using an immunoassay) to determine whether they produce a protein capable cf binding to anti-1 CAM-1 antibody.

The expression vectors of those cells which produce a protein capable of binding to anti-ICAM-1 antibody are then further analyzed to determine whether they express (anc thus contain) the entire ICAM-I gene, whether they express (and contain) only a fragment cf the ICAM-1 gene, or whether they excress (anG contain) a gene wr.ose product, though immunologically related to ICAM-1, is not ICAM-1. Although sucn an analysis may be performed by any convenient means, it is preferable to determine the nucleotide sequence of the ONA or cDNA fragment which had been cloned into the expression vector. Such nucleotide sequences are then examined to determine whether they are capable of encoding polypeptides having the same amino acid sequence as the tryptic digestion fragments of ICAM-1 (Table 5).

An expression vector which contains a ONA or cONA molecule whicn encodes the ICAM-1 gene may, thus, be recognized by: (i) the ability to direct the expression of a proteinswhich is capable of binding to anti -1CAM-1 antibody; anc (ii) the presence of a nucleotide sequence which is capable of encocing each of the tryptic fragments of ICAM-1. The cloned ONA molecule of such an expression vector may be removed from the exoression vector and isolated in pure form.

EXAMPLE 21 Functional Activities of Purified ICAM-1 In cells, ICAM-1 normally functions as a surface protein associated with the cell membrane. Therefore, the function of purified ICAM-I was tested after the molecule was reconstituted into artificial li 2 4^353 membranes (liposomes, or vesicles) by dissolving the protein in cetergent-solubilized licics, followed by the removal of the detergent by dialysis. I CAM-1 purified from JY cells and eluted in the detergent octylglucoside as descr::=: above was reconstituted into vesicles, and the ICAM-1 containing -.esicles were fused to glass covers 1 ips or plastic culture wells to allow the detection of cells binding to the protein.

Preparation of planar fne":ranes and plastic-bound vesicles Vesicles were prepare; by the method of Gay et al.. (J. Immunol. 136:2025 (1935)). 3rie;,y, egg phosphatidylcholine and cholesterol were dissolved in chlorc-':rm and mixed in a molar ratio of 7:2. The lipid mixture was dried t: a thin film while rotating under a stream of nitrogen gas, anc was the-, lyophilizad for 1 hour to remove all traces of chloroform. The lip-; film was then dissolved in 1% octylcluco-side/0.14 M NaCl/20 mM Tris (pH 7.2) to a final concentration of phosphatidylcholine of 0.1 mM. Approximately 10 of purified ICAM-1, or human glycopnorin (Sig~a Chemical Co., St. Louis, MO) as a control membrane glycoprotein, was added to each ml of dissolved lipids. The orotein-lipid-detergent solution was then dialyzed at 4'C against 3 changes of 200 volumes cf 20 mM Tris/0.14 M NaCl, pH 7.2, and one change of HESS.

% Planar membranes were prepared by the method of Brian et al.. Proc. Natl. Acad. Sci. 81:5159 (1984). Glass coverslips (II mm in diameter) were boiled for 15 minutes in a 1:5 dilution of 7X detergent (Linbro), washed overnight in distilled water, soaked in 70% et'nanol, anc air dried. An £0 ii 1 drop of vesicle suspension containing either ICAM-1 or glycophorin was placed in the bottom of' wells in a 24-well cluster plate, and the prepared glass coverslips were gently floated on top. After 20-30 minutes inegation at room temperature, the wells were filled with HBSS, and the coverslips were inverted to bring the planar membrane face up. The wells were then washed extensively with HESS to remove unbound vesicles.. The planar membrane surface was never exposed to air. 24^85 3 In the course of experiments with planar membrane fused to glass surfaces, vesicles containing ICAM-1 were found to bind directly to the plastic Surface of multi-well tissue culture plates, and retain functional activity as 'evidenced by specific cell binding. Such vesicles are hereinafter referred to as "plastic-bound vesicles" (?SV) since the nature of the lipic vesicles bound to the plastic has not been deter-inec. Plastic-bound vesicles were prepared by adding 30 ^1 of vesicle suspension directly to the bottom of wells in 96-we11 tissue culture trays (Falcon), followed by incubation and washing as described for pianar membranes.

Cell adh.es'on assays Cell a -e s i c n assays using planar membranes or pi astic-bounc vesicles were both cone in essentially the same way, except that the cell nurr.be-s anc volumes for P5V assays were reduced to one-fifth that used in planar membrane assays.

T-lymcr.ccytes from normal controls and a Leukocyte Adhesion Deficiency (LAD) patient whose cells fail to express LFA-I (Anderson, D.C. et a'.. J. Infect. Pis. 152:663 (1985)) were prepared by culturinc ceripheral blocd mononuclear cells with 1 ^g/ml Concanavalin-A (Con-A) in RPMI-15-0 plus 20% FCS at 5 x 10^ cells/ml for 3 days. The cells were then washed twice with RPMI and once with 5 mM methyl-alpha-0-manncpyrar.cside to remove residual lecbin from the cell surface. The cells were grown in RPMI/20% FCS containing 1 ng/ml recombinant IL-2, and were used between 10 and 22 days after the initiation of culture.

To detect cell binding to planar merr.Dranes or PBV, Con-A blasts, the T-lympnorr.a S'KW-3, and the EBV-transformed B-lymphoblastoid cell lines JY (LFA-1 positive) and LFA-1 deficient lymphoblastoid cell line (BEN) (derived from patient 1, Springer, T.A. et al.. J. Exoer. Mec. 160:1901-1913 (1S25) were radiolabeled by incubation of 1 x 10^ cells in 1 ml cf RPMI- 1640/10% FCS with 100 nCi of Na^CrO* for 1 hour at 37'C. followed by four washes with RPMI-1640 to remove unincorporated label. In monoclonal antibody blocking experiments, cells or plastic-bound vefcles were pretreated with 20 fig/ml of purified antibody in 2 4 40 RPMI-1640/10% FCS at 4"C for 30 minutes, followed by 4 washes to remove unbound antibccy. It. experiments on the effects of divalent cations on cell binding, the cells were washed once with Ca'"*", Mg^-free BBSS plus 10% dialyzed FCS, and CaCl and MaCl were added to the indicated concentrations. In all experiments, cells and planar membranes or P3V were pre-ecuiibrated at the appropriate temperature (4*C, 22"C, or 37*C) in the appropriate assay buffer.

To measure cell binding to purified ICAM-1, ^-Cr-labeled cells (5 x 103 E5V-traroformants in planar membrane assays; 1 x 103 E5V-transforma.nts or SKW-3 cells, 2 x IO3 Con-A blasts in PSV assays) were centrifuged for 2 minutes at 25 x g onto planar memoranes or P5V, followed by :r.cubation at 4*c, 22'C, or 37*C for one hour. After incubation, u-'pounc cells were removed by eight cycles of filling and aspiration w:t.~. buffer pre-equi 1 ibrated to the appropriate temperature. Bound cells -era quanlitatec by solubilization of well contents with 0.1 N NaOH/ri Triton X-100 and counting in a gamma counter. Percent cell binding was determined by dividing cpm from bound cells by input cell-associates cpm. In planar membrane assays, input cpm were corrected for the ratio of the surface area of coverslips compared to the surface area of the culture wells.

In tnese assays, E3V-trans formed B-lymphobl astoid cells, S'KW-3 T-lymphoma eel's, anc Con-A T-lymphoolasts bound specifically to ICAM-1 in artificial membranes (Figures 11 anG% 12). The binding was specific since the ce'ls bound very poorly to control planar membranes or vesicles containing equivalent amounts of another human cell surface glycoprotein. glycopnorin. Furthermore, LFA-1 positive E3V-transformants and Con-A blasts bound, while their LFA-1 negative counterparts failed to bind to any. significant extent, demonstrating that the bincing was dependent on the presence of LFA-1 on the cells.

Both the specificity of cell binding and the dependence on cellular LFA-1 were confirmed in monoclonal antibody blocking experiments (Figure 13). The binding of JY cells could be inhibited by S7% when the ICAM-l-containing PSV were pretreated with anc.i-ICAM-1 monoclonal antibocy RRPretreatment of the cells with the same antibody hac 0;2'.=3 2 4 4 0 5 3 little effect. Conversely, the anti-LFA-1 monoclonal antibody TS1/18 inhibited binding by 96%, but only when the cells, but not the PSV, were pretreated. A control antibody TS2/9 reactive with LFA-3 (a different lymphocyte surface antigen) had no significant inhibitory effect when either cells or PSV were pretreated. This experiment demonstrates that ICAM-1 itself in the artificial membranes, and not some minor contaminant, mediates the observed cellular adhesion and that the adhesion is dependent on LFA-1 on the binding cell.

The binding of cells to ICAM-1 in artificial membranes also displayed two other characteristics of the LFA-1 dependent aahesion system: temperature dependence and a requirement for divalent cations. As shown in Figure 14, Con-A blasts bound to ICAM-1 in PBV most effectively at 37*C, partially at 22*C, and very poorly at 4"C. As shown in Figure 15. the binding was completely dependent on the presence of divalent cations. At physiological concentrations, Mg^~ alone was sufficient for maximal cell binding, while Ca^"*" alone produced very low levels of binding. However, Mg^"*" at one-tenth of the normal concentration combined with Ca^r was synergistic and produced maximal binding.

In summary, the specificity of cell binding to purified ICAM-1 incorporated into artificial membranes, the soecific inhibition with monoclonal antibodies, and the temperature and divalent cation requirements demonstrate that ICAM-1 is a specific ligand for the LFA-1-dependent adhesion, system. v EXAMPLE 22 Expression of ICAM-1 and HLA-DR in Allergic and Toxic Patch Test Reactions Skin biopsies of five normal individuals were studied for their expression of ICAM-1 and HLA-OR. it was found that while the endothelial cells in some blood vessels usually expressed ICAM-1, there was no ICAM-1 expressed on keratinocytes from normal skin. No staining for HLA-OR on any keratinocyte from normal skin biopsies was observed. The kinetics of expression of ICAM-1 and class II antigens were then o ?. A 0 £ 4 "f ^ studied on cells in biopsies of allergic and toxic skin lesions. It was found that one-half of the six subjects studied had keratinocytes which expressed ICAM-l, four hours after application of the hapten (Table 10). There was an increase in the percentage of individuals expressing ICAM-1 on their keratinocytes with time of exposure to the hapten as well as an increase in the intensity of staining indicating more I CAM-1 expression per keratinocyte up to hours. In fact, at this time point a proportion of keratinocytes in all bioosies stainec positively for ICAM-1. At 72 hours (2- hours after tne hapten was removed), seven of the eight subjects still had ICAM-1 expressed on their keratinyocytes while the expression of ICAM-1 on one subject waned between 48 and 72 hours.

TABLE 10 Kinetics of Induction of ICAM-I and HLA-OR on Keratinocytes from Allergic Patch Test Biopsies Time After Patch No. of ICAM-1 HLA-OR ICAM-1& Application (h) Biopsies Only Only HLA-OR Normal Skin Allergic Patch Test 4 5 8 9 24 8 48b 8 72 8 3a 0 0 3 0 0 7 0 0 0 3 6 0 1 aSamoles were considered as positive if at least small clusters cf keratinocytes were stained. bAll patches were removed at this time point. n c 7 • - - 2 4 4 3 Histologically, the staining pattern for ICAM-1 on keratinocytes from biopsies taken four hours after application of the haoten was usually in small clusters. At 43 hours, ICAM-1 was expressed on the surface of the majority of the keratinocytes, no difference being seen between the center and periphery of the lesion. The intensity of the staining decreased as the keratinocytes approached the stratum corneum. This was found in biopsies taken from both the center anc the periphery of the lesions. Also at this time point, the patch test was positive (infiltration, erythema and vesicles). No difference in ICAM-1 expression was observed when different haptens were aopliec on sensitive individuals. In addition to keratinocytes, ICAM-L was also expressed on some mononuclear cells and endothelial cells at the site of the lesion.

The expression of HLA-OR on keratinocytes in the allergic skin lesions was less frequent than that of ICAM-1. None of the subjects studied had lesions with keratinocytes that stained positively for HLA-OR up to 24 hours after the application of the hapten. In fact, only four biopsy samples had keratinocytes that expressed HLA-OR anc no biopsy had keratinocytes that was positive for HLA-OR and not ICAM-1 (Table 10).

In contrast to the allergic patch test lesion, the toxic patch test lesion induced with croton oil or sodium lauryl sulfate had keratinocytes that displayed little if'"any ICAM-1 on their surfaces at all time points tested (Table 11). In fact, at 43 hours after the patch application, which was the optimum time point for ICAM-1 expression in the allergic patch test subjects, only cne of the U toxic patch test subjects had keratinocytes expressing ICAM-1 in their lesions. Also in contrast to the allergic patch test biopsies, there was no HLA-OR expressed on keratinocytes of toxic patch test lesions.

These data indicate that ICAM-1 is expressed in immune-based inflammation and not in toxic based inflammation, and thus the expression of ICAM-1 may be used to cistinguish between immuno basec and toxic based inflammation, such as acute renal failure in kicney transolant patients where it is difficult to cetermme whether failure r L is due to rejection or nephrotoxicity of the immunosuppressive theraoeutic agent. Renal biopsy and asse-sment of upregulation of [CAM-1 expression will allow differentiation of immune based rejection and non-immune based toxicity reaction.

TABLE 11 Kinetics of Induction of ICAM-1 and HLA-OR on Keratinocytes from Toxic Patch Test 3iopsies Time After Patch No. of ICAM-1 HLA-CR ICAM-1S Aoolication (h) Biopsies Only Only HLA-CR 4 4 0 0 0 3 la 0 0 24, 3 10 0 43° 14 1 0 0 72 3 10 0 aSamples were considered as positive if at least small clusters of keratinocytes were stained. bAll patches were removed at this time point. v EXAMPLE 23 Expression of ICAM-1 and HLA-OR in Benign Cutaneous Diseases Cells from skin biopsies of lesions from patients with various types of inflammatory skin diseases were studied for their expression of ICAM-1 and HLA-OR. A proportion of keratinocytes in biopsies of allergic contact eczema, pemphigoid/pemphigus ana licnen planus expressed ICAM-1. Lichen planus biopsies showed the most intense staining with a pattern similar to or even stronger than that seen ir. the ^3-hour allergic patch biopsies (Table 12) Consistent with 24485 3 results seen in the allergic patch test, the most intensive ICAM-1 staining was seen at sites of heavy mononuclear cell infiltration. Furthermore, 8 out of the 11 Lichen planus biopsies tested were positive for HLA-OR expression on keratinocytes.

The expression of ICAM-I on keratinocytes from skin bioosies of patients with exanthema and urticaria was less pronounced. Only four out of the seven patients tested with these diseases hac keratinocytes that expressed I CAM-i at the site of the lesion. HLA-OR expression was only found on one patient anc this 'was in conjunction with ICAM-1.

Endothelial cells ana a proportion of the mononuclear cell infiltrate from all the benign infl amatory skin ciseases tested expressed ICAM-1 to a varyinc extent.

TABLE 12 Expression of ICAM-1 and HLA-DR on Keratinocytes from Benign Cutaneous Diseases No. of ICAM-1 HLA-OR I CAM -1i Diagnosis Cases Only Only HLA-DR Allergic Contact Eczema 5 3a % 0 2 Lichen Planus 11 3 0 S Pemphigoid/ Pemphigus 2 2 0 0 Exanthema 3 2 0 0 Urticaria 4 10 1 aSamples were considerec as positi/e if at least small clusters of keratinocytes were stained. 0921S3 q ; / L 4 ^ EXAMPLE 24 Excression of ICAM-1 on Keratinocytes of Psoriatic Skin Lesions The expression of ICAM-1 in skin biopsies from 5 patients with psoriasis were studied before the initiation and periodically during a course of Pl'V- treatment. Biopsies were obtained from 5 patients with classical psoriasis confirmed by histology. Biopsies were taken secuentially refore ana during indicated time cf PUVA treatment. PUVA was given 3 t: 4 times weekly. Biopsies were taken from the periphery cf the psGr:;:;c plaques in five patients anc, in addition biopsies were taken f-:~ clinically normal skin in four of the patients.

Fresh ski- biopsy specimens were frozen and stored in liquid nitrogen. Six micron cryostat sections were air dried overnight at room temcerat.re, fixed in acetone for 10 minutes and either stained immediately c wrapped in aluminum foil anc stored a: -SO*C until staining.

Staining was accomplished in the following manner. Sections were incubated witn monoclonal antibodies and stained by a three stage immunoperoxidase method (Stein, H., et. al.. Adv. Cancer Res £2:57-1*7, (1SS4)), using a diaminobenzidine 'H2O2, substrate. Tonsils and lymph nodes were used as positive control for anti-ICAM-1 and HLA-DR staining. Tissue stained in the absence of primary antibody were negative controls.

The monoclonal antibodies against HLA-DR were purchased from 3ecton Cickinson (Mountainview, California). The monoclonal anti-1 CAM -1 antibody was RS-5-DS. Peroxidase-conjugated rabbit anti-mouse Ig and peroxidase-conjucated swine anti-rabbit Ig were purchased from DAKAPATTS, Cccenhagen, Denmark. Diaminobenzidine-tetrahydrochloride were obtained from Sigma (St. Louis. Mo.).

The results of the study show that the endothelial cells in some blood vesse': express ICAM-1 in both diseased and norma' skin, but trre intensity c"" staining and the number of blood vessels expressing ICAM-1 092133 2 4 ' 3 was increased in the psoriatic skin lesions. Moreover, the pattern of expression of ICAM-1 in keratinocytes of untreated psoriatic skin lesions from the five patients varied from only small clusters of cells staining to many keratinocytes being stained. During the course of PUVA treatment, the ICAM-1 expression on Z of the patients (patients Z and 3) shewed marked reduction which preceded or was concurrent with clinical remission (Table 13). Patients I, ^ and 5 had decreases and increases of ICAM-1 expression during the PUVA treatment which correlated to clinical remissions and relapses, respectively. There was no I CAM-1 expression on keratinocytes from normal skin before or after PUVA treatment. This indicates that PUVA does not induce ICAM-1 on keratinocytes from normal skin.

Of note was the observation that the csnsity of the mononuclear cell infiltrate correlated with the amount of ICAM-1 expression or, keratinocytes. This pertained to both a decreased number of mononuclear cells in lesions during PUVA treatment when ICAM-1 expression also waned anc an increased number of mononuclear cells during PUVA treatment when ICAM-1 expression on keratinocytes was more prominent. Endothelial cells and derma; mononuclear cells are also I CAM-1-positive. In clinically normal skin, ICAM-1 expression was confined to endothelial cells with no labelling of keratinocytes.

The expression of HLA-CR on keratinocytes was variable. There was no HLA-OR positive biopsy that was not ^1 so ICAM-1 positive.

In summary, these results show that before treatment, ICAM-1 expression is pronounced on the keratinocytes and correlate to a dense mononuclear cellular infiltrate. During PUVA treatment a pronounced decrease of the ICAM-1 staining is seen to parallel the clinical improvement. Histologically the dermal infiltrate also diminished. When a clinical relapse was obvious during treatment, the expression of ICAM-1 on the keratinocytes increased, as well as the density of the dermal infiltrate. When a clinical remission was seen during treatment, there was a concurrent decrease in ICAM-1 staining on tne keratinocytes as well as decrease in the dermal infiltrate. Thus the expression of ICAM-1 on keratinocytes corresponded to the density of Z V' J tr.e mononuclear cellular infiltrate of the dermis. These data show that clinical response to PUVA treatment resulted in a pronour.ied decrease of ICAM-1 expression on keratinocytes parallel to a more mccerata decline of the mononuclear cells. This indicates that ICAM-1 expression on keratinocytes is responsible for initiating and maintaining the dermal infiltrate and that PUVA treatment down regulates ICAM-1 which in turn mitigates the dermal infiltrate and the inflammatory response. The data also indicates that there was variable HLA-OR expression on keratinocytes during PUVA treatment.

The expression of ICAM-1 on keratinocytes of psoriatic lesions correlates with the clinical severity of the lesion as well as with the size of the dermal infiltrate. Thus ICAM-1 plays a central role in psoriasis and inhibition of its expression and/or mnibition of its i-.teraction with the CD 13 complex on mononuclear cells will be an effective treatment of the disease. Furthermore, monitoring ICAM-1 expression on keratinocytes will be an effective tool for diagnosis and prognosis, as well as evaluating the course of therapy of psoriasis. s l.W? pC ~- 2 4 4 8 5 3 - 87 -TABLE 13 Sequential ICAM-1 Expression by Keratinocytes in Psoriatic Skin Lesions (PS) and Clinically Normal Skin (N) Before and During PUVA Treatment patient no.

Time before 12 3 4 and during PUVA treatmen PS PS N PS N PS N PS N 0 1 day - 1 week - - r-r - + 0 2 weeks -- r - r - T 3 weeks — 0 4 weeks r - - - t*I ★ •k -6 weeks - • 0 7 weeks (++) (+) T-TT * ★ weeks (*) Many positive keratinocytes +- A prooosition of positive keratinocytes r Focal positive keratinocytes (r) Very few scattered positive keratinocytes No positive staining s * Clinical remission 0 Clinical relapse EXAMPLE 25 Expression of ICAM-1 and HLA-OR in Malignant Cutaneous Diseases Unlike lesions from benign cutaneous conditions, the expression of ICAM-1 on keratinocytes from malignant skin lesions was much more variable (Table U). Of the 23 cutaneous T-cell lymphomas studied, ICAM-1 positive keratinocytes were identified in only H cases. There A3-.1.W? C921S3 Z 4 ^ 8 5 3 was a tendency for keratinocytes from biopsies of mycosis fungoiaes lesions to lose their ICi'M-l expression with progression of the disease to more advanced stages. However, ICAM-1 expression was observed on a varying proportion of the mononuclear cell infiltrate from most of the cutaneous T cell lymphoma lesions. Among the regaining lymphomas studied, four of eight had keratinocytes that expressec ICAM-I. Of the 29 patients with malignant cutaneous diseases examined, 5 had keratinocytes that expressed HLA-OR without expressing ICAM-1 (Table U).

TABLE 14 Expression of ICAM-1 and HLA-DR on Keratinocytes from Malignant Cutaneous Diseases No. of ICAM-1 HLA-DR I CAM-11 Di agnosi s Cases Only Only HLA-CR CTCL, MFI 8 la 0 * CTCL, MFII -111 1 2 CTCL, SS 3 1 0 2 CTCL, Larae Cel 1 2 0 2 0 C8CL 2 0 0 i 4 Leukemia Cutis 3 1 1 Histiocytosis X 1 0 0 r- s aSamples were considered as positive if at least small clusters cf keratinocytes were stained.

EXAMPLE 25 Effect of Anti-ICAM-1 Antibodies on the Proliferation of Human Peripheral Blood Mononuclear Cells Human peripheral blood mononuclear cells are induced to proliferate by the presence and recognition of antigens or mitogens. Certain-molecules, such as the mitogen, concanavalin A, or i.-.e T-cel 1 -hinci~.g 2 k'' 8 5 3 antibody 0KT3, cause a non-specific pro!iferation of peripheral blooc mononuclear cells to occur.

Human perioheral blood mononuclear cells are heterogeneous in that they are comcosec of subpopulations of cells which are capable of recognizing soecific antigens. When a peripheral blood mononuclear cell which is capable of recognizing a particular specific antigen, encounters tne antigen, the proliferation of that subpopulation of mononuclear cell is induced. Tetanus toxoid and keyhole limpet hemocyanin are examples of antigens which are recognized by subDopulations of peripheral mononuclear cells but are not recognizee by all peripheral mononuclear cells in sensitized individuals.

The ability of anti-1 CAM -1 monoclonal antibody R6-5-D6 to inhibit prol iferative responses of human peripheral blood mononuclear cells in systems known to reauire cell-cell achesions was tested.

Peripheral blocc mononuclear cells were purified on Ficoll-Paque (Pharmacia) gradients as per the manufacturer's instructions. Following collection of the interface, the cells were washed three times with RPMI 15-0 medium, and cultured in flat-bottomed 96-well microtiter plates at a concentration of 20° cells/ml in RPMI I5-10 medium supplemented with 10% fetal bovine serum, 2mM glutamine, anc gentamicin (50 ^c/ml).

Antigen, either the T-cell mitogen, concanavalin A (0.25 /xg/ml); the T-cel 1 -binding antibody, CKT3 (0.001 ^c/ml); keytiole limpet hemocyanin (10 g/ml) or tetanus toxoid (1:100 dilution from source) were added to cells which were cultured as described above in either the presence or absence of anti-ICAM antibody (R5-5-D6; final concentration of 5 g/ml).

Cells were cultured for 3.5 days (concanavalin A experiment), 2.5 days (0KT3 experiment), or 5.5 days (keyhole limpet hemocyanin and tetanus toxoid experiments) before the assays were terminated.

Eighteen hours prior to the termination of the assay, 2.5 pCi of thymidine was added to the cultures. Cellular proliferation was assayed by measuring the incorporation of thymidine into DMA by tn& peripheral blood mononuclear cells. Incorporated thymidine was collected anc counted in a liauid scintillation counter (Merluzzi et oc-: 2 448 5 - SO al ., J. Immunol. 139:156-153 (1987)). The results of these experiment are shown in Figure 15 (concanavalin A experiment), Figure 17 (CKT3 experiment), Figure 18 (keyhole limpet hemocyanin experiment), anc Figure 19 (tetanus toxoid experiment).

It was found that anti-ICAM-1 antibody inhibits proliferative responses to the non-specific T-cell mitogen, ConA; the non-specific T-ce 11 associated antigen, OKT-3; and the specific antigens, keyhole limpet hemocyanin ana tetanus toxoid, in mononuclear cells. The inhibition by anti-ICAM-1 antibody was comparable to that of anti-LFA-1 antibody suggesting that ICAM-1 is a functional ligand of LFA-1 and that antagonism of ICAM-1 will inhibit specific defense syst=~ responses.

EXAMPLE 27 Effect of Anti-ICAM-1 Antibody on the Mixed Lymphocyte Reaction As discussed above, ICAM-1 is necessary for effective cellular interactions during an immune response mediated through LFA-1 -dependent cell adhesion. The induction of ICAM-1 during immune responses or inflammatory disease allows for the interaction cf leukocytes with each other and with endothelial cells.

When lymphocytes from two unrelated^indivduals are cultured in eacr. others presence, blast transformation and cell proliferation of the lymphocytes are observed. This response, of one population q-lymphocytes to the presence of a second population of lymphocytes, is known as a mixed lymphocyte reaction (MLR), and is analogous to tr.e response of lymphocytes to the addition of mitogens (Immunology T'-e Science of Self-Nonself Discrimination. Klein, J., John Wiley & Sons, NY (1982), pp 453-453).

Experiments were performed to determine the effect of anti-ICAM monoclonal antibodies on the himan- MLR. These ' experiments wore conducted as follows. Peripheral blood was obtained from norT.a"., healthy donors by veniouncture. The blood was collected in heparin nee 2 4485 91 - tubes and diluted 1:1 at room temperature with Puck's G (GI6C0) balances salt solution (BSS). The blood mixture (20 ml) was layers.-1 over 15 ml of a Ficoll/rlypaque density gradient (Pharmacia, density = 1.078, room temperature) and centrifuged at 1000 x g for 20 minutes. The interface was then collected and washed 3X in Puck's G. The cells were counted on a hemacytometer and resuspended in RPMI-15-iO culture medium (GI SCO) containing 0.5'i of gentamicin, 1 mM L-glutamina (GIBCO) and 5*i heat inactivated (5o*C, 30 min.) human AS sera (Flow Laboratories) (hereafter referred to as RPMI-culture medium).

Mouse anti-ICAM-1 (R6-5-05) was used in these experiments. All monoclonal antibodies (prepared from ascites by Jackson ImmunoResearch Laboratories, Soston, MA) were used as purified IgG preparations.

Peri:neral blood mononuclear cells (PBMC) were cultured in medium at 6.25 x IO3 cells/ml in Linbro round-bottomed microliter plates (=76-013-05). Stimulator cells from a separate donor were irradiated at 1000 R ana cultured with the responder cells at the same concentration. The total volume per culture was 0.2 ml. Controls included responder cells alone as well as stimulator cells alone. The culture plates were incubated at 37*C in a 5?i CC^-humidified air atmosphere for 5 days. The wells were pulsed with 0.5 /iCi of tritiated thymidine (^HT) (New England Nuclear) for the last 18 hours of culture. In some cases a two-way MLR was performed. The protocol was the same except that the second donor's cells were not inactivated by irradiation.

The cells were harvested onto glass fiber filters using an automated multiple sample harvester (Skatron, Norway), rinsing with water and methanol. The filters were oven dried and counted in Aquasol in a Beckman (LS-3801) liquid scintillation counter. Results are reported as the Mean CPM t standard error of 6 individual cultures.

Table 15 shows that purified anti-ICAM-1 monoclonal antibodies inhibited the MLR in a dose dependent manner with significant suppression apparent at 20 ng/ml. Purified mouse IgG had little or no suppressive effect. Suppression of the MLR by the anti-ICAM-1 monoclonal antibody occurs when the antibody is added within the first 2- hours of cultures (Table 16).

TABLE 15 1 ,•48 5 3 Effect of Anti-ICAM-I Antibody on the Cns-Way Lymphocyte -.eacticn Ressor.cer Cells2 Stimulator Cells-3 Ant:bocvc -HT Incorporation (CPM' - .71 IgG rrucG - mlgG (10 ( 0 ( 0 0 I • */ r* ~ 1 . - »c) .02 U) , 35, 42, 5CC 3 t • w i ~ ♦ r> * *s ~ i ^ ^ r :,::j : 1,2-5 t • i crv, ( -"'I — - R6-5 + R5-5 + R5-5 -06 -D6 -Do (10.0 lie) ( O.i ug) ( 0.03 ]i<z) 8. 15 23. 250 1-2 o «« U "* : 520 = 8:3 : I.7S0 ! 1 3' 1 . >> (:::i; (32%) a. Responder cells (6.25 X 10V^) b. Stimulator Cells (5.2: x lOV^i. irradiated at 10CCR) c. Purified Monoclonal Antibody to ICAM-1 (R5-5-D6) cr purifiec rrcuse IgG (mlgG) at final concentrations (uc/.-nl). d. Mean £ S.E. of 5-5 cultures, numbers in parentheses indicate oerce".: inhibition of MLR ^ V :o 2-4 8 5 3 TA3LE 16 Time of Addition of Anti-1CAM-1 Ra S:3 -coitions0 ^HT Incorporation (C?M) Time of Addition of Med;u~ or -nt'bccv Cay 0 Day 1 Cay Z -edium 205d : 14 *76 = 122 2*7 ; 75 r "cdium 189 ± 15 nce nc •i- - ~edium 1,860 r 515 nd na + + .Tiedium 41,063 t 2,940 45,955 t 2,947 50,943 ; 3,072 + + R5-5-D6 17,731 ; 1,293 33,409 r 1,531 47,303 r 2.0S9 (57%)r (15%) (7%) a. Responder cells (6.25 x 10^/ml) b. Stimulator Cells (c.25 x 103/ml, irradiated at 1CQ0R) c. Culture Medium or Purified Monoclonal Antibody tc ICAM-1 (R6-5-06) at 10 £ic/-i were added on day 0 at 24 hour intervals d. Mean : S.E. of 4-6 cultures e. nd = not cone v f. Percent Inhibition In summary, the ability of antibody against ICAM-1 to inhibit the MLR shows t"5t ICAM-1 monoclonal antibodies have t.ierapeutic utility in acute graft rejection. ICAM-1 monoclonal antibodies also have therapeutic utility in related immune mediated disorders Gependent on LFA-1/ICAM-I regulated cell to cell interactions.

The experiments described here show that the aodition of monoclonal antibodies to ICAM-1 inhibit the mixed lymphocyte reaction (MLR) wner-added durir.c the first 24 hours of the reaction. Furthermore, ICAM-1 2 4 4 8 5 3 becomes upregulated on human peripheral blood monocytes during ]_n vitro culture.

Furthermore, it was found that ICAM-1 is not exoressed on resting human peripheral blood lymphocytes or monocytes. ICAM-1 is up regulated on the monocytes of cultured cells alone or cells co-cu1turec with unrelated doner cells in a mixed lymphocyte reaction using conventional flow cytometric analyses. This up regulation of ICAM-1 on monocytes can be usee as an indicator of inflammation, particularly if ICAM-1 is expressed on fresh monocytes of individuals with acute or chronic inflammation.

ICAM-1's specificity for activated monocytes and the ability of antibody against ICAM-1 to inhibit an MLR suggest that ICAM-1 monoclonal antibodies may have diagnostic and therapeutic potential in acute graft rejection and related immune mediated disorders requiring cell to cell interactions.

EXAMPLE 23 Synergistic Effects of the Combined Administration of Anti-ICAM-1 and Anti-LFA-1 Antibodies As shown in Example 27, the MLR is inhibited by anti-ICAM-1 antibody. The MLR can also be inhibited by the anti-LF^-1 antibody. In order to determine whether the combined administration of anti-1CAM-1 and anti-LFA-1 antibodies would have an enhanced, or synergistic effect, an MLR assay (performed as described in Example 27} was conducted in the presence of various concentrations of the two antibodies.

This MLR assay revealed that the combination of anti-ICAM-1 and anti-LFA-1, at concentrations where neither antibody alone dramatically inhibits the MLR, is significantly more potent in inhibiting the MLR response (Table 17). This result indicates that therapies which additionally involve the administration of anti-ICAM-1 antibody (or fragments thereof) and anti-LFA-1 antibody (of fragments thereof) have the capacity to provide an improved anti-inflammatory therapy. Such an 2 4 4 8 improved theraoy permits the administration of lower doses of antibody than would otherwise be therapeutically effective, and has importance in circumstances where high concentrations of individual antibodies induce an anti-idiotypic response.

TABLE 17 Effect of Various Doses of Anti-ICAM-i and (R3.1) Anti-LFA-1 on Mixed Lymphocyte Reaction Of /o Inhibition Concentration (ug/ml) Ant * - LrA-i 0 .004 Anti -.02 ICAM ,-1 .1 (R5-5-D6) . 5 2.5 C.O 0 7 31 54 69 70 0.0008 1 7 28 48 62 71 C.004 0 13 50 64 72 0.02 29 38 64 75 84 86 0.1 92.5 90 91 92 92 92 0.5 93 90 90 V 92 93 91 EXAMPLE 29 Additive Effects of Combined Administration of Sub-optimal Doses Anti-ICAM-1 and Other Immunosuppressive Agents in the MLR As shown in Example 28, the MLR is inhibited by combinations of anti-ICAM-1 and anti-LFA-1 antibodies. In order to determine whether the ccmbinec administration of anti-ICAM-1 ana other immunosuppressive 2 ■' 4 8 5 3 agents (such as dexamethasone. azathioprine, cyclosporin A or steroids (such as, for example, prednisone, etc.) would also have enhanced effects, MLR assays were performed using sub-optimal concentrations (i.e concentrations which would be lower than the optimal concentration at which the acent alone would be provided to a subject) of R6-5-06 in conjunction with other immunosuppressive agents as per the protocol in Examole 27.

The data indicate that the inhibitory effects of R5-5-06 are at least additive with the inhibitory effects of suboptimal doses of dexamethasone (Table IS), Azathioprine (Table 19) and cyclosporin A (Table 20). This implies that an t i-1CAM-1 antibodies can be effective in lowering the necessary doses of known immunosuppressants, thus reducing their toxic sice effects. In using an anti-ICAM-1 antibody (or a fragment thereof) to achieve such immunosuppression, it is possible to combine the administration of the antibody (or fragment thereof) with either a single additional immunosuppressive agent, or with a combination of more than one additional immunosuppressive agent. 2 4 4 8 5 3 TABLE 13 Effect of Anti-ICAM-1 and Dexamethasone on the Human MLR Group Inhibitor (ng/ml) 3HT Incorporation (CPM) 9/ /0 inhibition Medi a Stimulators (S) Responders (R) R x S 155 io i 4,461 34,129 ~ R x S R5-5-D6 (3) "7 ? ^ 23 R x S Dex (50) 14 (133- 59 R x S R6-5- D6 (3) - Dex (E3) 7,759 77 Dex: Qexametnasone TABLE IS Effect of Anti -ICAM-1 and Azithioor ine on the Hu: ?,an MLR Grouo s Inhibi tor (nc/ml) 3HT Incorporation (CPM1 9f .'0 Inhibition Medi a Stimulators (S) Resoonders (R) R x' S - 78 174 3,419 49,570 - R x S R6-5-D6 (8) 44,374 11 R x S Azathioprine (!) 42,710 l4 R x S R5-5-06 (S) - Azathioprine (I) 2^,246 31 2 4 4 8 5 3 TABLE 20 Effect of Anti-ICAM-1 and Cyclosporin A on the Human MLR Group Inhibitor (n a / TI 1) 3H7 Incorporation fCPMl Of to Inhibition Medi a Stimulators Responders R x S (S) (R) - 37 206 987 31,54C - R x S R5-5-D5 (3) ,232 17 R x S CyA (10) 23,517 R x S R5-5-06 (8) - CyA (1C) 19,204 39 CyA: Cyclosporin A EXAMPLE 20 Effect of Anti-ICAM-1 Antibody in Suppressing the Rejection of Transplanted Allogeneic Organs v, In order to demonstrate the effect of anti-ICAM-1 antibody ir, suppressing the rejection of an allogeneic transplanted organ. Cynomolgus monkeys were transplanted with allogeneic kidneys according to the method described by Cosimi et al. (Transplant. Proc. j_3;499-5C2 (1981)) with the modification that valium and ketamine were used as anesthesia.

Thus, the kidney transplantation was performed essentially as follows. Heterotropic renal allografts were performed in 2-5 <z Cynomolgus monkeys, essentially as described by Marquet (Marquet n al.. Medical Primatolocv. Part II, Basel, Karger, p. 125 (1972)! af:=-induction of anesthesia with van-m and ketamine. End-to-s;:= 2 4^8 anastomoses of donor renal vessels cn a patch of aor:a or vena cava were constructed using 7-0 Prolene suture. The dcnor ureter was spatulated and implanted into the bladder by the extrsvesical approach (Tagucni, Y., et al .. in Oausset et al . (ads.). '<*'■ Advances in Transplantation. Baltimore, Williams 1 Wilkins, p. 393 (1953)). Renal function was evaluated by weekly or biweekly serum creatinine determinations. In addition, frequent allograft biopsies were ootainec for histopathologic examination and co.nolete autopsies were performec on all nonsurviving recipients. In most recipients, bilateral nephrectomy was performed at the time of transplantation anc subsecuent uremic death was considered the end point of allograft survival. In some recipients, unilateral native nephrectomy anc contra!ateral ureteral ligation were performed at the time of transplantation. When allograft rejection occurred, the ligature on the autologous ureter was then removed resulting in restoration of normal renal function anc the opportunity to continue immunologic monitoring of the recipient animal.

Monoclonal antibody R5-5-D5 was administered daily for 12 days starting two days prior to transplant at a dose of 1-2 mc/kg/day. Serum levels of creatinine were periodically tested to monitor rejection. The effect of anti-ICAM-1 antibody on the immune system's rejection of the allogeneic kidneys is shewn in Table 21. 2 4 4 8 5 3 TABLE 21 R6-5-06 Activity in Prolonging Renal Allograft Survival in Prophylactic Protocols in the Cynomolcus Monkeva Days of Survival/ Monkey Dose of R6-5-D6 (mg/kg) Post-Treatment Control 1 3 Control 2 - i 1 Control 3 - 11 Control 4 - Control 5 - 9 Control 5 - Ml 5 1.0 Ml 9 1.0 7° Ml 7 1.0 M25 1.5 29 M23 1.0 llc M27 2.0 34 M7 0.5 22 Mil 0.5 M10 0.5 22 MS 0.5 25d Monkeys were given R6-5-05 for 12 consecutive days starting at 2 days prior to transplantation. s Causa of daath is unknown. Thera was evidence of latent malaria.

Died of kidney infarct.

Still living as of August 15, 1938.

The results show that R5-5-D5 was effective in prolonging the lives of monkeys receiving allocenic kidney transplants.

I 4 4 0 5 - 101 -EXAMPLE 31 Effect of Anti-ICAM-1 Antibccy in Suppressing Acute Rejection of Transplanted Organs In order to snow that anti-ICAM-I antibody is effective in an acute model of transplant rejection, R5-5-D5 was also tested in a therapeutic or acute kidney rejection ~odel. In this model, mcn.<ey kidneys were transplanted (using the protocol described in Example 30) and given perioperative!./ 15 mg/kg cyclosporin A (CyA) i .m. until stable renal function was achieved. The dose of CyA was then recuced biweekly in 2.5 mg/kg increments until rejection occurred as indicated by a rise in blood creatinine levels. At this point. R6-5-05 was administered for 10 days and tne lengtn of survival was -onitorea. It is important to note that in tnis protocol, the dose cf CyA remains suboptimal since it does not change once t.ne acute rejection episode occurs. In this model historical c:ntrols (N=5) with no antibody rescue survive 5-1-1 days from the onset of the rejection episcce. To date, six animals were tested using ".5 - 5 - Do in this protocol (Table 22). Two of these animals are still surviving (M12. 31 days and M5, 47 days following the administration of Ro-5-06). Two animals lived 38 and 55 days following initiations cf R5-5-D6 therapy and two animals died from causes other than acute rejection (one animal diec of CyA toxicity and the other died while being given R5-5-D6 under^anesthesia). This model more closely approximates the clinical situation in which R6-5-06 would be initially use:. n / f 2 4 ^ < - 1C2 -TABLE 22 R5-5-D6 Activity in Prolonging Renal Allograft Survival in Therapeutic Protocols in the Cynomdgus Monkey2 Days of Survival/ Monkey Day of Rejection Episode^ Post-Treatment Control sc l-i-98 5-14 M24 41 38 .

M21 34 4d M3 41 53 MS 12 ll5 M12 37 >31r_ M5 >47" Monkeys were given 1-2 mg/kg cf R5-5-D6 for 10 consecutive cays following onset of rejection.

Day at which creatinine levels increased as a result of reduction of CyA cosace and R5-5-D6 therapy started.

Five animals were tasted using the therapeutic protocol describec above exceot that there was no rescue therapy. Days of survival/post treatment represents days of survival once creatinine levels started to rise.

Died while under anesthesia. Creatinine levels were low.

Oied of CyA toxicity. Creatinine'level s were low.

Still living as of August 15, 1983.

EXAMPLE 32 Genetic Construction anc Expression of Truncated Derivatives of ICAM-1 In its natural state, ICAM-1 is a cell membrane-bound protei containing an extracellular region of 5 immunoglobulin-1ike domains, transmembrane domain, ana a cytoplasmic domain. It was desirable t construct functional derivatives of iCAM -1 lacking the transmemcran domain and/or the cytcclasmic domain in that a soluble, secretes for O f. k ^ L L-i : ' of ICAM-1 could be generated. These functional derivatives were constructed by oligonucleotic*-directed mutagenesis of the ICAM-1 gene, followed by expression in monkey cells after transfection with the mutant gene.

Mutations in the ICAM-1 gene resulting in amino acid substitutions and/or truncated derivatives were generated by the method of Kunkel, 7., (?**oc. Natl. Acad. Sci. (U.S.A.) 82:^38-492 (1985)). ICAM-1 cDNA prepared as described above was digested with restriction endonucleases Sal 1 and K:n 1, and the resulting l.S kb DNA fragment was subcloned into the plastic vector CDM8 (Seed, B. et al.. Proc. Natl. Acad. Sci. ;"J. S. A. 1 :3255-3359 ( 1987)). A dut*, una* strain of E. col i (3W313/P3) was then transformed with this construct, designated oCDl.SC. A single-strand uraci1-containing template was rescued from the transformants by infection with the helper phage R408 (Stratagene^). Mutant ICAM-1 cDNAs were then generated by priming a second stranc synthesis with an oligonucleotide possessing mismatched oases, anc subseauent transformation of a una" host (MC10S1/P3) with the resulting heteroduplex. Mutants were isolated by screening for newly created endonuclease restriction sites introduced by the mutant oligonucleotide. The mutant ICAM-1 protein was expressed by transfection of Cos-7 cells with the mutant DNA in the eukaryotic expression vector CDM8 using standard DEAE-Dextran procedures (Selders, R.F. et al.. in: Current Protocols in 'Molecular Biolocv (Ausubel, F.M. et al.. eds.) pages 9.2.1-9.2.6 (1987)).

A truncated functional derivative of ICAM-1 lacking the transmembrane and cytoplasmic domains, but containing the extracellular region possessing all 5 immunoglobulin-1ike domains was prepared. A 30 bp mutant oligonucleotide (CTC TCC CCC CGG TTC TAG ATT GTC ATC ATC) was used to transform the codons for amino acids tyrosine (Y) and glutamic acid (E) at positions 452 and 453, respectively, to a phenylalanine (F) and a translational stop codon (TAG). The mutant was isolated by its unique X':a 1 restriction site, and was designated Y^r/p ( To express the mutant protein, COS cells were transfected with three mutuant subclones (t2. *7, and #8). Three days after 0 f A " K L L-< - ,J transfection with the three mutant subclones, culture supernates and eel: lysate were analysed by immunoprecipitation with anti-ICAM-1 -onoclonal antibody RR1/1 and SOS-PAGE. ICAM-1 was precipitated from the culture suoernates of cells transfected with mutant subclones = 2 and tS, but not from detergent lysates of those cells. The molecular weight of ICAM-i found in the culture supernate was reduced aDcroximately 5 !<c relative to the membrane form of ICAM-1, which is consistent with the size predicted from the mutant DNA. Thus, Znis functional derivative of ICAM-1 is excreted as a soluble protein. In contrast, IC.-M-I was not immunoprecipitated from control culture sucernates of cells transfected with native ICAM-1, demonstrating that the membrane ""orm of ICAM-1 is not shed from Cos ceils. Futnermore. no ICAM-1 was i.mmunocracipi tated from either culture supernates or cell lysates from negative control mock-transfected cells.

The truncates ICAM-1 secreted from transfected cells was purified by imnunoaffinity chromatography with an ICAM-1 specific antibody (R5-5-C6) anc testes for functional activity in a cell binding assay. After purification in the presence of the detergent octylglucoside, preparations containing native ICAM-1 or the truncated, secreted form were diluted to a final concentration of 0.25% octylglucoside (a concentration below the critical micelle concentration of the detergent). These preparations of ICAM-1 were allowed to bind to the surfaces of plastic 96-well plates (Nunc), to produce ICAM-1 bound to a solid-phase. After washing out unbound material, approximately 75-80% anc 83-88% of SKW-3 cells bearing LFA-1 on their surface bound soecifically to the native and to the truncated forms of ICAM-1, respectively. These data demonstrate that the secreted, truncated soluble ICAM-1 functional derivative retained both the immunological reactivity and the ability to mediate ICAM-1 dependent adhesion which are characteristic of native ICAM-1.

A functional derivative of ICAM-1 lacking only the cytoplasmic domain was prepared by similar methods. A 25 bp oligonucleotide (TC AGC ACG TAC CTC TAG AAC CGC CA) was used to alter the codon for a--.no ac:d 475 ('<] to a TAG translational stoo codon. The mutant was 2 4 A e 5 3 105 - designated Y^/TAG. Immunoprecipitation analysis and SOS-PAGE of Cos calls transfected with the mutant detected a membrane bound form of ICAM-1 with a molecular weight approximately 3 kd less than native ICAM-1. Indirect immunofluorescence of the mutant-transfected Cos cells demonstrated a punctate staining pattern similar to naive ICAM-1 expressed on LPS-stimulated human endothelial cells. Finally, cells transfected with the mutant ONA specifically bound to purified LFA-1 on plastic surfaces in a manner similar to Cos cells transfected with native ICAM-1 ONA (Table 23).

TABLE 23 Ability of Cells Expressing ICAM-1 or a Functional Derivative of ICAM-1 to Bind LFA-1 % of Cells Expressing ICAM-1 that Bind LFA-1 in the Presence of: TRANSFECTION No Antibody RR1'1 Mock 0 0 Native ICAM-1 20 C Yd76/TAG 20 0 EXAMPLE 33 MAPPING OF ICAM-1 FUNCTIONAL DOMAINS Studies of ICAM-1 have revealed that the molecule possesses 7 domains. Five of these domains are extracellular (domain 5 being closest to the cell surface, domain 1 being furthest from the cell surface), one domain is a transmembrane domain, and one domain is cytoplasmic (i.e. lies within the cell). In order to determine which domains contribute to the ability of ICAM-1 to bind LFA-1, epitope mapping studies may be used. To conduct such studies, different deletion mutants are prepared and characterized for their capacity to :.3- I UP 2 4 n 5 bind to LFA-1. Alternatively, the studies may be accomplished usinc anti-ICAM antibody known to interfere with the caoacity of ICAM-1 to bind LFA-1. Examples of such suitable antibody include RRI/1 (Rothlein, R. et a].., J. Immunol. 137:1270-127'! (1936)), R5.5 (Springer, T.A. et al., U.S. Patent Application Serial "o. 07/250,4-5), LB - 2 (Clark, E.A. et al .. In: Leukocyte Typing I (A. Bernard, et al.. Eds.), Sprincer-Verlag pp 339-346 (1SS-)), or CL203 (Staunton, Q.E. et al ., Cell 55:849-3S3 (1989)).

Deletion mutants of ICAM-1 can be created by any cf a variety of means. It is, however, preferable to produce such mutants via site directed mutagenesis, or by other recomoinant means (such as by constructing ICAM-1 expressing gene sequences in whicr. sequences that encode particular protein regions have been celetec. Procedures which may be adapted to produce such mutants are well known in the art. Using such procedures, three ICAM-1 deletion mutants were preoarec. The first mutant lacks amino acid residues F185 through P2S4 (i.e. deletion of domain 3). The second mutant lacks amino acid residues P284 through R451 (i.e. deletion of domains - and 5). The third mutant lacks amino acid residues after Y475 (i.e. deletion of cytoplasmic domain). The results of such studies indicate that domains 1, 2, and 3 are predominantly involved in ICAM-1 interactions with anti-ICAM-1 antibody ana LFA-1.

% EXAMPLE 34 EFFECT OF MUTATIONS IN ICAM-1 ON LFA-1 SINC IMG The ability of ICAM-1 to interact with ana bind to LFA-1 is mediated by ICAM-1 amino acid residues which are present in domains 1 of the ICAM-1 molecule (Figures 8, 9 and 10). Such interactions are assisted, however, by contributions from amino acids present in domains I and 3 of ICAM-1. Thus, among the preferred functional derivatives cf the present invention are soluble fragments of the ICAM-I molecule whicn contain domains I, 2, and 3 of ICAM-I. More care soluble 2 4 A l -■ -> fragments of the ICAM-1 molecule which contain domains 1 and 2 of 1CAM-1. Most preferred are soluble fragments of the ICAi'-l molecule which contain domain 1 of ICAM-1. Several amino acid residues within the first ICAM-1 domain are involved in the interaction of ICAM-1 and LFA-1. Substitutions of these amino acids with other amino acids alter the ability of ICAM-I to bind LFA-1. These amino acid residues and the substitutions are shown in Figure 25. Figure 25 shows the effects of such mutations on the ability of the resulting mutant ICAM-1 molecule to bind to LFA-1. In Figures 23-25, residues are described with reference to the one letter code for amino acids, followed by tne position of the residue in the ICAM-1 molecule. Thus, for example. "£90" refers to the glutamic acid residue at position SO of ICAM-1. Similarly, "ESOV" refers to the dipeptide composed of the glutamic acic residue at position SO and the valine residue at position SI. The substitution sequence is indicated to the right of the slash (",/") mark. The V4, R13, Q27, Q58, and 060S61 residues of ICAM-1 are involved in LFA-1 binding.

Replacement of these amino acids altered the capacity of ICAM-1 to bind to LFA-1. For example, replacement of V4 with G results in the formation of a mutant ICAM-1 molecule which is less able to bine to LFA-1 (Figure 25). Replacement of the R13 residue of ICAM -1 with E leads to the formation of a mutant molecule with substantially less capacity to bind LFA-1. (Figure 25). Replacement of the Q53 residue of ICAM-1 with H leads to the formation of a mutant molecule having a substantially normal capacity to bind LFA-1 (Figure 25). Reolace.^ent of the DSOS residues of ICAM-1 with KL leads to the formation of a mutant molecule having substantially less capacity to bind LFA-1 (Figure 25).

Glycosylation sites in the second domain are also involved in LFA-1 binding (Figure 23). Replacement of N103 with K, or A155N with SV, results in the formation of a mutant ICAM-1 molecule whici is substantially incapable of binding LFA-1. In contrast, replacemeit of the glycosyl ation site N175 with A did not appear to substantially effect the capacity of the mutant ICAM-1 to bind LFA-1.

A3--. 1. WP I 4 A *— Mutations in the third ICAM-1 domain die not appreciably altar I CAM -1 - LFA-1 binding (Figure 24).

EXAMPLE 35 MULTIMERIC FORMS OF ICAM-1 WITH INCREASED SIOLCGICAL HALF-LIFE AFFINITY AND CLEARANCE ABILITY Chimeric molecules are constructed in wnich domains 1 and 2 of ICAM-1 are attached to the hinge region of the immunoglobulin heavy chain. Preferred constructs attach the C-terninus of ICAM-1 domain 2 to a segment of the immunoglobulin heavy chain gene just N-terminal to the hinge region, allowing the segmental flexibility conferred by the hinge region. Tne ICAM-I domains 1 and 2 will :hus replace the Fab fragment of an antiwOdy. Attachment to heavy cnains of the IgG class and production of animal cells will result in the production of a chimeric molecule. Procuction of molecules containing heavy chains derived from IgA or IcM will result in production of molecules of higher multimericy containing from 2 to 12 ICAM-1 molecules. Co-expression of J-c'nain gene in the animal cells producing the ICAM-1 heavy chain chimeric molecules will allow proper assembly of IgA and IgM multimers resulting predominantly in IgA molecules containing 4 to 6 ICAM-i molecules anc in the case of IgM containing approximately 10 ICAM-1 molecules. These chimeric molecules may have several abvantages. First, Ig molecules are designed to be long lasting in the circulation and this may improve bi ol ogi cal rial f-1 i fe.

Furthermore, the multimeric nature of these engineered molecules will allow them to interact with higher avidity with rhinovirus as well as with cell surface LFA-1, depending on the therapeutic context, and thus greatly decrease the amount of recombinant protein which needs to be administered to give an effective dese. IgA and IgM are highly glycosylated molecules normally present in secretions in mucosal sites as in the nose. Their highly hydrophilic nature helps to keep bacteria and viruses to which they bind out in the mucosa, preventing attachment to cells and creventing crossing of tne epithelia" ceil membrane 2 4l'-f' - 10s - barrier. Thus, they may have increased therapeutic efficacy. IgM and in particularly IgA are stable in mucosal environments and they may increase the stability of the ICAM-1 constructs. If such an ICAM-1 functional derivative is administered in the blood stream, it may also increase biological half-life. IgA does not fix complement and thus would be ideal for applications in which this would be deleterious. If IgG H chain chimerics are desired, it would be possible to mutate regions involved in attachment to complement as well as in interactions with Fc receptors.

EXAMPLE 35 GENERATION Or ICAM-I MUTANTS 01 iaonucleotide-di rected mutscene si s The coding region of an ICAM-1 cDNA in a 1.8 kb Sall-Kpnl fragment, was subcloned into the expression vector COMB (Seed, B. et al.. Proc. Natl. Acad. Sci. (U.S.A.) 84:3365-3363 (1937)). 8ased on the method of Kunkel, T.t (Proc. Natl. Acad. Sci. (U.S.A.) 82:488-492 (1985)) anc modifications of Staunton 0. et al . (Staunton, O.E. et al.. Cel' £2.:925-933 (1988)), this construct (pCQl.8) was used to generate i single strand uracil containing template to be used in oligonucleotide-directed mutagenesis. s Briefly, E. col i strain XS127 was transformed with pCD1.8. Single colonies were grown in one ml of Luria Broth (LB) medium (Difco) witr. 13 Mg/ml ampicillin and 8 /ig/ml tetracycline until near saturation. 100 ill of the culture was infected with R4C8 helper phage (Strategene; at a multiplicity of infection (MOI) of 10, and 10 ml of LB medium wit", ampicillin and tetracycline was added for a 16 hr culture at 3/*C. Following centri fugation at 10,000 rpm for one minute, and 0.22 infiltration of the supernatant, the phage suspension was used to infect E. col i BW313/P3 which was then plated on LB agar (Difco) plate: supplemented with ampicillin and tetracycline. Colonies were picket, crown in 1 ml LB medium with ampicillin and tetracycline to near A / f- ^ 2 4f- - no - saturation anc infected with helper phage a: MO I of 10. Culture volume was then increased to 250 ml and the cells were cultured overnight. Single strand DNA was isolated by standard phage extraction.

Mutant oligonucleotides were phospnoryiated ana utilized with the pCOl.S template in a second strand synthesis reaction (Staunton, D.E. et al.. Cell 52:925-933 (1983)).

Transfection COS cells were seeded into 10 cm tissue culture plates such that they would be 50% confluent by 15-24 hrs. COS cells were then washed once with TBS anc incubated for 4 hrs with 4 oil RPMI containing 10% Nu sera (Collaborative) 5 fig/ml c'nloroquine, 3 jig of mutant plasmid ana 200 Mg/^1 DEAE-cextran sulfate. Cells were then washed wit 10% DMSO/PBS follcwec by PSS and cultured 15 hrs in culture mecia. Culture media was replaced with fresh media and at 43 hrs post transfection (OS cells were susoenced by trypsin/EOTA (Gibco) treatment and divided into 2, 10 cm plates as well as 24 well tissue culture plates for HRV binding. At 72 hrs cells were harvested from 10 cm plates with 5 mM EDTA/HBSS anc processed for adhesion to LFA-1 coated plastic and immunofluorescence.

LFA-1 and HRV binding N LFA-1 was purified from SKW-3 lysates by immunoaffinity chromatography an TS2/<1 LFA-1 mAb Sepahrose and eluted at pH 11.5 in the presence of 2 mM McC12 and 1% octylcucoside. LFA-1 (10 fig per 200 /il per 6-cm plate) was bound to bacteriological Petri dishes by diluting octylglucoside to 0.1% in PBS (phosphate buffered saline) with 2 mM MgCl? and overnight incubation at -'C. Plates were blocked with 1% BSA (bovine serum albumin) and storec in PBS containing 2mM MgCl?. 0.2% BSA, 0.025'/. azide, and 50 ug/ml gentamycin.

^Cr-labelled COS cells in PBS containing 5% FCS (fatal calf serum), 2 mM MgC12» 0.025% azide (buffer) were incubated with or without 5 A3-.1•V>'P C92::; 2 4 ^5 - ill - /ic/rol RRI/1 and R5.5 in LFA-1 coated microtiter platas at 25*C for 1 hour. Non-ac'nerent cells were removed by 3 washed with buffer. Adherent cells were eluted by the addition of EOTA to 10 mM and 7-counted.

RESULTS Anti - ICAM-i antibodies such as RRI/1, R5.5, L3-2, or CL203 have been identified. If these antibodies are capable of inhibiting ICAM-1 function, they must be capable of binding to a particular site in the ICAM-1 molecule which is also important to the ICAM-1 function. Tnus, by preparing tne above-described deletion mutants of ICAM -1, anc determining the extent to which the anti-ICAM-1 antibodies can bind to the deletion, it is possible to determine whether the deletea domains are imcortan*. for function.

ICAM-1 is an integral membrane protein, of which the extracellular domain is predicted to be composed of 5 Ig-like C-dcmains. To identify domains involved in binding LFA-1, domain 3 and domains 4 and 5 (carboxyl terminal) were deleted by ol igonucleotide-directec mutagenesis and tasted functionally following expression in COS ceils. In addition, the entire cytoplasmic domain was deleted to ascertain its potential influence on ICAM-1 interactions. As expected the cytoplasmic domain deletion, Y476/* demonstrated no loss of RRI/1, R5.5, L3-2 and CL203 reactivity whereas, deletion of domain 3, F185-R451, resulted in a decrease and loss of CL203 reactivity, respectively (Figure 20). Thus, the CL203 epitope appears to be located in domain -whereas RRI/1, R5.5 and L3-2 appear to be located in the 2 aminc-terminal domains.

All 3 deletion mutants demonstrate wild type levels of adherence to LFA-1 (Figure 21). Amino acid substitutions in predicted 3-tums in domains 1, 2 and 3 were also generated and functionally tested following excression in COS cells. The R5.5 epitODe was thus localizes to the secuence E111GGA in domain 2 and may also involve E39 in domain 1 whereas RP.1/1 and L3-2 are both dependent on R13 in domain 1 (Figure Z 4 4 P 22). In accition, RRI/1 binding is decreased by mutations in the sequence D71GQS. Mutations eliminating N-linked glycosylation sites at N103 and N155 result in decreased RRI/1, R5.5 and L3-2, LFA-1 HRV binding. These mutations appear to effect processing such that ICAM-1 cixers are generated.

Other mutations in domain 2 or 3 did not result in altered LFA-1 achesion (Figures 23 and 24). The amino acids in domain 1, R13 and 060 bctn are involved with binding LFA-1 (Figure 25).

Thus, LFA-1 and HRV binding appears to be a function of the amino terminal Ig-like domain of ICAM-1. Figure 25 shows an alignment of ICAM amino terminal domains.

While the invention has been described in connection with specific e-oadiments thereof, it will be understood that it is caoable of further mccifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, tr.e principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth as follows in the scope of the appended claims.

Claims (8)

30 What we claim is:

1. A pharmaceutical composition comprising a) an anti-inflammatory agent selected from: an antibody capable of binding to ICAM-1; a fragment of an antibody, said fragment being capable of binding to ICAM-1; ICAM-1; a functional derivative of ICAM-1 as herein defined; and a non-immunoglobulin antagonist of ICAM-1; and b) at least one immunosuppressive agent.

2. A pharmaceutical composition according to claim 1 wherein said immunosuppressive agent is selected from dexamethasone, azathioprine and cyclosporin A.

3. An anti-inflammatory agent for use in the preparation of a pharmaceutical composition to be used together with an immunosuppressive drug for treating inflammation resulting from a response of the specific defence system, said anti-inflammatory agent being as defined in claim 1.

4. An anti-inflammatory agent according to claim 3 wherein said immunosuppressive drug is selected from dexamethasone, azathioprine and cyclosporin A.

5. An anti-inflammatory agent for use in the preparation for a pharmaceutical composition for treating inflammation resulting from a response of the specific defence system to be used together with a second agent chosen from: an antibody, capable of binding to LFA-1; a functional derivative of an antibody, said functional derivative being capable of binding to LFA-1; and a non- immunoglobulin antagonist of LFA-1; said anti- 114 inflammatory agent being as defined in claim 1.

6. A pharmaceutical composition or anti-inflammatory agent according to any one of the preceding claims wherein the anti-inflammatory agent is the antibody R6-5-D6.

7. A pharmaceutical composition according to claim 1, substantially as described with reference to any Example thereof.

8. An anti-inflammatory agent according to claim 3 or claim 5, substantially as herein described. DANA FARBER^ANCER INSTITUTE j!(£jSIW3LA ■ by their'Attorneys BALDWIN, SON & CAREY